Refined ChIP‐Seq Protocol for High‐Quality Chromatin Profiling in Solid Tissues Using the Complete Genomics/MGI Sequencing Platform

Abstract

The chromatin immunoprecipitation followed by sequencing (ChIP‐seq) assay is an instrumental and accurate method for understanding chromatin dynamics in eukaryotic cells. It provides critical insights into the regulation of gene expression and enables identification of regulatory elements, patterns of histone modifications, and chromatin states in health and disease conditions. Although cell cultures are great models to study molecular mechanisms associated with pathologies, studying tissues provides a physiologically native environment that reflects the cellular heterogeneity and spatial organization that are missing in an in vitro model. Several ChIP‐seq protocols have been published; however, performing ChIP‐seq in tissues remains a challenge in many settings due to the heterogeneity of tissues, complexity of cell matrices, low input material and intricacy of chromatin fragmentation and handling. Here, we present an optimized ChIP‐seq protocol for solid tissues, with a focus on colorectal cancer. In this article, we incorporate simplified and efficient procedures for tissue preparation, chromatin extraction, immunoprecipitation, and library construction. The refined protocols overcome common limitations related to tissue processing and allows for highly reproducible, sensitive, and scalable analysis of disease‐relevant chromatin states in vivo. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC.

Basic Protocol 1: Frozen tissues preparation

Basic Protocol 2: Chromatin immunoprecipitation from tissues

Basic Protocol 3: Library construction for DNA sequencing

Basic Protocol 4: DNA nanoballs preparation for the DNBSEQ‐G99RS sequencing platform and data quality control

INTRODUCTION

Mammalian tissues are complex structures with diverse cell types, where spatial organization contributes to the tissue's function through the regulation of gene expression landscape (Adler et al., 2023; Bissell et al., 1999; Parada et al., 2004; Pavlicev et al., 2024; Xavier Da Silveira Dos Santos & Liberali, 2019). Unlike analyses on homogeneous cell populations, examining chromatin structure in a tissue context provides insights into how gene regulation is shaped by the tissue organization and highlights particular regulatory mechanisms that might be concealed in cell line models, leading to a better understanding of tissue‐related chromatin dynamics in health and diseases (Chapski et al., 2019; Greene et al., 2015). However, working with tissues presents considerable technical challenges, including complexities related to cell heterogeneity and matrices density, low input, limited resolution, low reproducibility, and challenging data analysis and interpretation (Damodaran et al., 2015; Lee et al., 2023; Navani et al., 2022).

Chromatin immunoprecipitation followed by sequencing (ChIP‐seq) has emerged as a powerful method for interrogating protein‐chromatin interactions, mapping chromatin modifications across the genome, and decoding the regulatory landscapes of gene expression both in vitro and in vivo (Gade & Kalvakolanu, 2012; Mundade et al., 2014; Park, 2009; Xu et al., 2020). The ChIP‐seq method, when applied to tissue studies, allows to investigate chromatin dynamics in vivo while preserving chromatin architecture and signaling pathways within a tissue‐specific context (Furey, 2012; Hanssen et al., 2017; Yadav & Kundra, 2023). Although several studies and protocols have been published, performing ChIP‐seq in tissues remains a highly demanding and challenging method due to limitations associated with chromatin fragmentation and isolation from complex tissue matrices, while preserving protein–DNA interactions (Cayir et al., 2024; Cotney & Noonan, 2015; Perna & Alberi, 2019; Texari et al., 2021).

Here, we propose a refined ChIP‐seq approach for chromatin analysis in solid tissues, with a specific focus on colorectal cancer tissues. These protocols help overcome limitations due to the dense and heterogeneous nature of solid tissues. Through optimized procedures of tissue preparation, chromatin extraction, and immunoprecipitation, these protocols allow the preservation of tissue‐specific chromatin features and enhances output data quality. It also integrates library construction compatible with Complete Genomics/MGI sequencing platforms, which offers a cost‐effective and efficient sequencing alternative, particularly suitable for large cohort studies.

This article is organized into a series of four comprehensive Basic Protocols (Fig. 1). Basic Protocol 1 describes a simple method for the preparation of frozen tissue samples for ChIP application. It includes refined steps for mincing frozen tissues under cold conditions, followed by two proposed homogenization options. Basic Protocol 2 describes steps of tissue samples cross‐linking with formaldehyde and processing for chromatin extraction. Ensuring proper tissue handling and processing is critical to preserve chromatin integrity and minimize degradation. Next, the protocol describes the optimized ChIP workflow of cross‐linked samples with detailed steps of lysis, chromatin shearing, and immunoprecipitation, with emphasis on optimized buffer composition, shearing parameters, and washing steps, to minimize background noise and to enhance quality of ChIPed DNA, allowing a reproducible chromatin profiling in tissues. In Basic Protocol 3, we outline libraries construction procedures for Complete Genomics/MGI sequencing platform, including end‐repair and A‐tailing, adaptor ligation with MGI‐specific adaptors, and PCR amplification steps, with multi‐stage quality checkpoints. Finally, in Basic Protocol 4 we outline the procedure for DNA nanoballs (DNBs) preparation and sequencing, and we describe the data quality control (QC) output from the sequencer.

Figure 1.

Overview of refined ChIP‐seq protocol for high‐quality chromatin profiling in solid tissues using the Complete Genomics/MGI Sequencing Platform. Basic Protocol 1: Frozen tissues preparation. Frozen tissue mincing followed by homogenization using Dounce tissue grinder or gentleMACS Dissociator. Basic Protocol 2: Chromatin immunoprecipitation from tissues. Homogenized tissue samples are cross‐linked with formaldehyde and processed for chromatin shearing and ChIP. Basic Protocol 3: Library construction for DNA sequencing. Library construction procedures for Complete Genomics/MGI sequencing platform, including end‐repair and A‐tailing, adaptor ligation with MGI‐specific adaptors, PCR amplification, and circularization. Basic Protocol 4: DNA nanoballs preparation for the DNBSEQ‐G99RS sequencing platform and data quality control. DNA nanoballs (DNBs) preparation for DNBSEQ‐G99RS sequencing platform and data analysis. Created with BioRender.com.

NOTE: All protocols involving animals must be reviewed and approved by the appropriate Animal Care and Use Committee and must follow regulations for the care and use of laboratory animals. Appropriate informed consent is necessary for obtaining and use of human study material.

Basic Protocol 1. FROZEN TISSUES PREPARATION

This is a systematic, step‐by‐step protocol for preparing frozen tissue samples for the ChIP assay. It includes mincing and homogenization of frozen tissues under cold conditions. This protocol was optimized using two homogenization alternatives: a semi‐automated method using a gentleMACS Dissociator and a manual method using a Dounce tissue grinder. Depending on the sampling method, tissues might vary vastly in size, particularly human and animal biopsies (see Supporting Information, Fig. S1). This protocol addresses this variability, using standardized steps that are suitable for all tissue sizes and leading to consistent and reproducible output values.

Materials

• Frozen tissue samples, i.e., colorectal tumors and their adjacent normal tissues (non‐tumors) in this protocol

• 1× phosphate‐buffered saline (PBS) supplemented with protease inhibitors (see recipe), 4°C

• Biosafety cabinet (BSC)

• Ice bucket with ice

• Sterile Petri dishes

• Sterile scalpel blades

• Sterile Dounce tissue grinder, 7‐ml, pestle A (MilliporeSigma, cat. no. D9063) or gentleMACS Dissociator (Miltenyi Biotec, cat. no. 130‐093‐235) and gentleMACS C‐tubes

• 50‐ml conical tubes

• Refrigerated benchtop centrifuge for 50‐ml tubes, 4°C

• 1Take frozen tissue cryotubes from –80°C and put them directly on ice. Proceed immediately to next steps.
• 2
  Tissue preparation is performed in a biosafety cabinet (BSC).

  • a.
    Transfer the samples (on ice) to the BSC.
  • b.
    Place a Petri dish in the center of the ice bucket, and using a sterile scalpel, retrieve the tissue sample and place it in the center of the Petri dish.
  • c.
    Ensure that the Petri dish is firmly stable on the ice and will remain stationary while manipulating the tissue (Fig. 2, A1‐A3 and B1‐B3).
    It is recommended to measure the size of the tissue, and to keep an image for record.

Figure 2.

Experimental methods for tissue processing and homogenization using Dounce tissue grinder and gentleMACS Dissociator. (A) Representative images showing tissue homogenization performed with a Dounce grinder. Minced tissue is transferred to the Dounce grinder. The wall of the Dounce homogenizer is rinsed with 1× PBS supplemented with protease inhibitors to collect all tissue fragments at the bottom. The tissue fragments are homogenized by applying uniform strokes of the “A” pestle. The pink color is an indicator of a good homogenization. (B) Representative images of tissue homogenization using the gentleMACS Dissociator. Minced tissue is transferred to the C‐tube. The wall of the C‐tube is rinsed with 1× PBS supplemented with protease inhibitors to collect all tissue fragments at the bottom. The tube is inverted to allow the tissue fragments to settle into the cap (rotator‐stator unit). The C‐tubes are placed upside‐down on the gentleMACS Dissociator and the optimal predefined program is run.

• 3Mince the tissue sample with two scalpel blades on the Petri dish (on ice) until finely diced.
• 4Using both scalpels, collect the minced tissue and place it in Dounce tissue grinder or C‐tube for gentleMACS Dissociator.

Homogenization

• 5aTransfer the minced tissue to the 7‐ml Dounce grinder (on ice; Fig. 2, A4).
• 6aUse 1 ml of cold 1× PBS supplemented with protease inhibitors to rinse the walls of the grinder, ensuring that all tissue fragments are brought to the bottom (Fig. 2, A5).
• 7aShear the tissue by applying even strokes of the A pestle (8 to 10 times) as shown in Figure 2, A6.

  Following Dounce homogenization, the presence of some debris and clumps is expected, as connective tissue and fat may resist complete disruption.

  Be careful of splashing, spills, or glass breakage from excessive speed.

  Sink the Dounce grinder deeply into the ice to keep it cold while maintaining firm control on it by holding it tightly by the bulb.

  To prevent tissue warming, avoid holding the bottom of the grinder by hand.

• 8aPour 2 to 3 ml of cold 1× PBS supplemented with protease inhibitors into the Dounce homogenizer.
• 9aPour the contents of the Dounce into a new 50‐ml conical tube.
• 10aUsing 2 to 3 ml of cold 1× PBS supplemented with protease inhibitors, gradually rinse the Dounce homogenizer and transfer the wash into the 50‐ml tube from the previous step to ensure complete recovery of the tissue cells.
• 11aRepeat step 10a once.

Homogenization using gentleMACS Dissociator

• 5bTransfer the minced tissue to the C‐tube (on ice; Fig. 2, B4).
• 6bUse 1 ml of cold 1× PBS supplemented with protease inhibitors to rinse the walls of the C‐tube, ensuring that all tissue fragments are brought to the bottom of the tube (Fig. 2, B5‐B6).
• 7bTap the upside‐down C‐Tube on the lab bench to ensure that all material is in contact with the blade. The C‐tubes are placed upside‐down on the gentleMACS machine (Fig. 2, B7).
• 8bRun the “h_tumor_03.01” predefined program. This program is preconfigured on gentleMACS machines with parameters optimized for tissue homogenization and can be run without any additional modifications.

  Depending on the density and thickness of the tissue type, you may need to test multiple programs until achieving an optimal homogenization.

• 9bPour 2 to 3 ml of cold 1× PBS supplemented with protease inhibitors into the C‐tube.
• 10bPour the contents of the C‐tube into a new 50‐ml conical tube.
• 11bUsing 2 to 3 ml of cold 1× PBS supplemented with protease inhibitors, gradually rinse the C‐tube and transfer the wash into the 50‐ml tube from the previous step to ensure complete recovery of the tissue cells. Repeat this step once.
• 12Centrifuge tube(s) from step 11a or 11b for 10 min at 300 × g, 4°C.
• 13Discard supernatant by gently pouring to avoid losing material. Place the tube(s) on ice.

  Do note stop at this stage. Proceed immediately to Basic Protocol 2.

Basic Protocol 2. CHROMATIN IMMUNOPRECIPITATION FROM TISSUES

Here, we detail an optimized ChIP protocol for tissue samples from Basic Protocol 1. Using a 1% formaldehyde solution to cross‐link samples is optimal for ensuring high permeability and effective capture of a broad range of interactions, while preserving reversibility for downstream applications (Hoffman et al., 2015; Toth & Biggin, 2000). We propose an optimized composition of buffers for lysis, chromatin shearing, washes, and elution along with sample handling tips to help enhance ChIP efficiency. The conditions were systematically optimized for both tissue samples and cultured cells, and it was successfully tested on two different cell lines (K562 and HCT116). While sonication buffer composition is optimal for a broad range of tissues and cells, it is important to optimize the chromatin shearing conditions (power, duration, and number of cycles) according to the experimental design, sample type, and sonication device. This protocol was tested with a variety of antibodies and proved successful; for the purposes of this protocol, we present the ChIP results using the histone H3 lysine 27 acetylation (H3K27ac) antibody.

Materials

• Pellet in tube(s) from Basic Protocol 1

• 1% cross‐linking solution (see recipe), 30 ml per sample, room temperature

• 2.5 M glycine solution (see recipe)

• 1× PBS supplemented with protease inhibitors (see recipe), 4°C

• L₁‐buffer (see recipe), 4°C

• S₁‐buffer (see recipe), 4°C

• RNase A, 10 mg/ml (Thermo Fisher Scientific, cat. no. EN053)

• Proteinase K, 10 mg/ml (Thermo Fisher Scientific, cat. no. EO0491)

• PCR purification kit (e.g., Qiagen, cat. no. 28104)

• H₂O, nuclease‐free (Ambion, cat. no. AM9937)

• Qubit dsDNA BR assay kit (Invitrogen, cat. no. Q32850)

• Protein A Dynabeads (Invitrogen, cat. no. 10001D)

• 1× PBS supplemented with 0.1% Tween 20 (PBST) (see recipe), 4°C

• Anti‐H3K27ac antibody (Active Motif, cat. no. 39133)

• D₁‐buffer (see recipe), 4°C

• D₂‐buffer (see recipe), 4°C

• WA₁‐buffer (see recipe), 4°C

• WB₁‐buffer (see recipe), 4°C

• TE buffer, pH 8.0 (Invitrogen, cat. no. AM9849)

• E‐buffer (see recipe), room temperature

• 5 M NaCl (Current Protocols, 2006)

• UltraPure phenol:chloroform:isoamyl alcohol (25:24:1, v/v) (Invitrogen, cat. no. 15593031)

• Chloroform (Fisher Scientific, cat. no. C298‐500)

• 3 M sodium acetate (Current Protocols, 2006)

• GlycoBlue (Invitrogen, cat. no. AM9515)

• Ethanol (Fisher Scientific, cat. no. BP28184)

• Qubit dsDNA HS assay kit (Invitrogen, cat. no. Q32854)

• Tube rotator

• Refrigerated benchtop centrifuges for 1.5‐ and 50‐ml tubes

• P‐200 and P‐1000 pipettes and tips

• Temperature‐controlled space at 4°C for incubation (cold room or fridge)

• Bioruptor Pico device (Diagenode, cat. no. B01060010) with actively cooled water bath set to 4°C (or another sonication device)

• 1.5‐ml Bioruptor Pico microtubes with caps (Diagenode, cat. no. C30010016)

• 1.5‐ and 2‐ml DNA Lobind tubes

• Ice bucket with ice

• 0.5‐ml tubes

• Mini centrifuge

• Water baths, 37° and 65°C

• Qubit assay tubes (Invitrogen, cat. no. Q32856)

• Qubit 4 fluorometer (Thermo Fisher Scientific, cat. no. Q33238)

• Bioanalyzer or TapeStation (Agilent)

• Magnetic rack for Dynabeads

• Vortex mixer

• Paper towel

• 1Add 30 ml of 1% cross‐linking solution to the pellet in tube(s) from Basic Protocol 1, step 13.
• 2Place the tube(s) on a 50‐ml tube rotator and rotate for exactly 10 min at room temperature.
• 3Stop the cross‐linking by adding 3.7 ml of 2.5 M glycine to the tube(s) to achieve a final glycine concentration of 125 mM.
• 4Incubate on a tube rotator for 5 min at room temperature.
• 5Centrifuge 10 min at 300 × g, 4°C.
• 6Very carefully pour off the supernatant without disturbing the pellet.

  The pellet will be very fluffy due to the fat content. Discard it gently and carefully, as some material may be lost (Fig. 3A).

Figure 3.

Representative images of various views of “pellets” across early steps of Basic Protocol 2. (A) Centrifugation after cross‐linking (step 5, pellet indicated in black circle). (B) Centrifugation after wash with ice‐cold 1× PBS supplemented with protease inhibitors (at the end of step 10, pellet indicated in black circle). (C) Centrifugation after lysis (nuclei pellet indicated in black circle, step 17). (D) Sheared chromatin suspension post‐sonication. (E) Sheared chromatin suspension after centrifugation. The clear supernatant (black arrow) contains the sheared chromatin, while the white pellet contains nuclear debris.

• 7Wash the walls of the tube(s) by adding 3 ml of ice‐cold 1× PBS supplemented with protease inhibitors, 1 ml at a time. Be careful not to disturb the pellet.
• 8Centrifuge 10 min at 300 × g, 4°C.
• 9Discard supernatant using a P‐1000 pipette.
• 10Repeat steps 7 to 9 once.
• 11The pinkish color associated with blood vessels may fade depending on tissue content (Fig. 3B). Proceed immediately to step 12.

• 12Add 1 ml L₁‐buffer to the cell pellet from step 11.
• 13Centrifuge 5 min at 300 × g, 4°C.
• 14Discard supernatant using a P‐1000 pipette.
• 15Resuspend the pellet with 1 ml L₁‐buffer.
• 16Incubate on a tube rotator for 10 min at 4°C.
• 17Centrifuge 10 min at 800 × g, 4°C.
• 18Discard supernatant using a P‐1000 pipette. At this stage, the pellet is visibly whiter and predominantly contains nuclei (Fig. 3C).
• 19Add 1 ml S₁‐buffer to the pellet.
• 20Centrifuge 5 min at 800 × g, 4°C.
• 21Discard supernatant using a P‐1000 pipette.
• 22Repeat steps 19 to 21 once.
• 23Resuspend the pellet with 1 ml S₁‐buffer.

Chromatin shearing

The sonication step was optimized for shearing with the Diagenode Pico Bioruptor. If using the same device, please refer to the manufacturer's instructions to ensure the correct functioning of the Bioruptor and the proper shearing of chromatin. If you choose another sonication method, or you are using a different tissue/cell type, it is recommended to perform trial runs to determine the optimal parameters of chromatin shearing.

• 24Split the 1 ml sample suspension from step 23 into 150‐ to 200‐µl aliquots per Bioruption tube.
• 25Position the Bioruptor tubes inside the Bioruptor ring and place them into the actively cooled water bath set to 4°C.
• 26Set Bioruptor to 30 s ON, 30 s OFF, and run for 12 cycles. Following sonication, it is common for the samples to appear cloudy (Fig. 3D).
• 27Remove the tubes from the Bioruptor tube holder and centrifuge them for 10 min at 21,000 × g, 4°C, using a benchtop centrifuge.
• 28Transfer supernatant of all fractions from the same sample into a single 1.5‐ml LoBind tube and place it on ice (Fig. 3E).
• 29Aliquot 25 µl of sample for reverse cross‐linking and size control. Store remaining chromatin at 4°C for step 54.

  It is recommended to proceed to ChIP steps within 24 hr of sonication. If samples are not processed within a maximum of 36 hr, discard them. Do not freeze chromatin samples.

• 30Transfer 25‐µl aliquot of the sheared sample from step 29 to a 0.5‐ml tube.
• 31Add 1 µl RNase A (10 mg/ml). Mix by flicking the tube.
• 32Using the mini centrifuge, centrifuge 5 s at maximum speed, room temperature, and incubate at 37°C in a water bath for 30 min.
• 33Remove the tube from the water bath and centrifuge as in step 32.
• 34Add 1 µl proteinase k (10 mg/ml). Mix by flicking the tube.
• 35Centrifuge as in step 32, then reverse cross‐link by incubating at 65°C overnight (16 hr or more) in a water bath.
• 36Remove the aliquot from the water bath and centrifuge as in step 32.
• 37Purify DNA using PCR purification kit following manufacturer instructions. Finally, elute in 25 µl nuclease‐free water.
• 38Quantify the DNA from previous step using Qubit dsDNA BR assay kit, assay tubes, and fluorometer, following manufacturer instructions.
• 39Assess the fragment size distribution of DNA using a Tapestation/Bioanalyzer.

  Optimal sonication results in a narrow peak of DNA fragment size between 100 and 700 bp (Fig. 4).

Figure 4.

Assessment of fragment size distribution post‐sonication of tissue samples (Basic Protocol 2, step 39) using High Sensitivity D1000 ScreenTape kit, readout on Agilent 2200 TapeStation. (A) Gel image of sheared chromatin from the four tissue samples in Supporting Information, Figure S1. L = ladder. The concentration is relatively proportional to the size of the tissue samples (see Supporting Information, Fig. S1). (B) An electropherogram of sample T1 showing a sub‐optimal, yet “ChIPable” fragment distribution with a broad peak between 150 and 700 bp and a relatively extended shoulder between 700 and 1000 bp. (C) An electropherogram of sample T2 showing an optimal fragment distribution between 100 and 700 bp with a peak at 213 bp.

Beads preparation

This section covers bead‐antibody complex preparation. Magnetic beads are pre‐equilibrated in binding conditions. The beads are then incubated with the appropriate antibody (anti‐H3K27ac in this protocol) under gentle rotation to allow antibody immobilization on the beads. Proper bead preparation is critical for minimizing background and improving the overall signal‐to‐noise ratio of the assay.

• 40Remove Protein A Dynabeads from the refrigerator and mix thoroughly before use.
• 41
  Transfer an appropriate volume of Dynabeads (V_b) into a new 2‐ml LoBind tube, using the following formula to calculate the required amount.

  $$V_b=25\mu \mathrm{l}\times \left(N+1\right)$$ (1)

  Where V_b is the volume of beads and N is the number of samples.
• 42Add 1 ml of ice‐cold PBST to the beads. Mix by inverting 4 to 5 times and centrifuge as in step 32.
• 43Place the 2‐ml tube on the magnetic separation rack until the liquid becomes clear. Carefully remove and discard the liquid using a P‐1000 pipette.
• 44Repeat steps 42 and 43 twice.
• 45
  Remove the 2‐ml tube from the magnetic separation rack and add a volume (V_p) of ice cold‐PBST to the beads as follows.

  $$V_p=50\mu \mathrm{l}\times \left(N+1\right)$$ (2)

  Where V_p is the volume of ice‐cold PBST to be added to the beads and N is the number of samples.
• 46Gently mix the beads with ice‐cold PBST by flicking the tube until the beads are fully suspended. Do not vortex or shake vigorously.
• 47
  Add a volume of the antibody (V_a) to the 2‐ml tube of beads as follows.

  $$V_a=\left(\mathrm{volume}\mathrm{corresponding}\mathrm{to}1\mu \mathrm{g}\right)\times \left(N+1\right)$$ (3)

  Where V_a is the volume of antibody to be added to the tube from step 46 and N is the number of samples.
• 48Incubate the beads‐antibody mixture at 4°C for 3 hr on a rotator to allow beads–antibody complex formation.

• 49After 3 hr of incubation, take the beads–antibody tube from step 48, centrifuge as in step 32, and place the tube on the magnetic rack until the liquid becomes clear.
• 50Carefully remove and discard the supernatant using a P‐1000 pipette, ensuring not to disturb the beads.
• 51Remove the tube from the magnetic rack and add 1 ml of ice‐cold PBST to the tube. Mix by gently inverting 4 to 5 times.
• 52Centrifuge as in step 32, and place the tube on the magnetic rack until the liquid becomes clear. Carefully remove and discard the liquid using a P‐1000 pipette.
• 53
  Remove the tube from the magnetic rack and add a volume of D₁‐buffer (V_D1) to the tube of beads‐antibody. Mix by gently flicking the tube until the beads are completely mixed. Do not vortex or shake vigorously.

  $$V_{D1}=200\mu \mathrm{l}\times \left(N+1\right)$$ (4)

  Where V_D1 is the volume of D₁‐buffer and N is the number of samples.
• 54Remove the chromatin sample in step 29 from the refrigerator. Pipette 800 µl of the chromatin sample(s) in new 1.5‐ml DNA LoBind tube(s).

  Save the remaining amount of chromatin sample(s) in a refrigerator to be used later for input(s) DNA.

• 55Dispense 200 µl of beads–antibody mix from step 53 to the 800 µl sample tube(s) from step 54.
• 56Incubate the chromatin–beads–antibody mixture tube(s) on a rotator at 4°C overnight.

• 57Take the tube(s) from step 56 (containing “beads–antibody–chromatin” complexes), centrifuge as in step 32, and place the tube(s) on the magnetic separation rack until the liquid becomes clear.
• 58Carefully remove and discard the liquid using a P‐1000 pipette. Do not disturb the beads.
• 59Remove the tube(s) from the magnetic rack and gently add 1 ml D₂‐buffer to the tube(s).
• 60Rotate the tube for 5 min at 4°C.
• 61Centrifuge as in step 32, and place the tube on the magnetic separation rack until the liquid becomes clear.
• 62Carefully remove and discard the liquid using a P‐1000 pipette. Be careful not to disturb the beads.
• 63Repeat steps 59 to 62 once.
• 64Remove the tube from the magnetic rack and gently add 1 ml WA₁‐buffer to the tube(s).
• 65Rotate the tube for 5 min at 4°C.
• 66Centrifuge as in step 32, and place the tube(s) on the magnetic separation rack until the liquid becomes clear. Carefully remove and discard the supernatant using a P‐1000 pipette.
• 67Repeat steps 64 to 66 once.
• 68Remove the tube from the magnetic rack and add 1 ml WB₁‐buffer to the tube(s).
• 69Rotate the tube for 5 min at 4°C.
• 70Centrifuge as in step 32, and place the tube on the magnetic separation rack until the liquid becomes clear. Carefully remove and discard the supernatant using a P‐1000 pipette. Do not disturb the beads.
• 71Repeat steps 68 to 70 once.
• 72Remove the tube from the magnetic rack and add 1 ml TE buffer to the tube.
• 73Rotate the tube for 5 min at 4°C.
• 74Centrifuge as in step 32, and place the tube on the magnetic separation rack until the liquid becomes clear. Carefully remove and discard the supernatant using a P‐1000 pipette. Do not disturb the beads.
• 75Repeat steps 72 to 74 once.

• 76To elute immunoprecipitated chromatin, remove tube(s) in step 75 from magnetic rack, add 200 µl E‐buffer to the tube(s) and incubate for 10 min in a water bath set at 65°C.
• 77Centrifuge 2 min at 21,000 × g, 4°C.
• 78Place the tube(s) on the magnetic separation rack until the liquid becomes clear.
• 79Carefully collect the liquid using a P‐200 pipette and transfer it to a new 1.5‐ml DNA LoBind tube(s).
• 80Repeat steps 76 to 79 once and transfer the liquid to the same 1.5‐ml sample tube(s) in step 79.

  At the end of elution step, the total eluted volume of ChIPed sample is 400 µl.

• 81Save the sample(s) on ice until step 84.

  Storing samples for an extended period has not been tested at this stage; therefore, its effect on the quality of the ChIPed DNA is unknown. It is recommended to proceed directly to DNA extraction.

• 82From the remaining non‐ChIPed chromatin solution (from step 54), take 80 µl of chromatin sample and put it in a new 1.5‐ml DNA LoBind tube. This tube will serve as 10% input sample.
• 83Add 320 µl E‐buffer (final volume of input will be 400 µl). Mix by pipetting up and down 4 to 5 times.

• 84Take ChIPed DNA‐tube(s) from step 81 and Input tube(s) from step 83. To each tube, add 16 µl of 5 M NaCl to a final concentration of 200 mM.

  Do not mix content of tubes. ChIPed DNA and Input DNA should always be treated separately.

• 85Briefly vortex the tubes, then centrifuge as in step 32, and incubate for 4 hr at 65°C in a water bath.
• 86After 4 hr incubation, centrifuge tubes as in step 32, and add 1 volume of phenol/chloroform/alcohol isoamyl (25:24:1) to each tube.
• 87Vortex tubes at high speed for 10 s.
• 88Centrifuge 2 min at 21,000 × g, 4°C.
• 89Using a P‐200 pipette, carefully transfer the upper phase of each tube to a separate, new 1.5‐ml DNA LoBind tube.
• 90Add 2 volumes of chloroform to each tube.
• 91Vortex tubes at high speed for 10 s.
• 92Centrifuge 2 min at 21,000 × g, 4°C.
• 93Using a P‐200 pipette, carefully transfer the upper phase of each tube to a separate, new 1.5‐ml DNA LoBind tube.
• 94Add 1/10 volume of 3 M sodium acetate to each tube.
• 95Add 1 µl GlycoBlue.
• 96Add 2 volumes of cold 100% ethanol. Shake tubes vigorously.
• 97Incubate for 1 hr at –80°C.

  Stopping point: Incubation at –80°C for 1 hr could be replaced by incubation at –20°C overnight.

• 98Remove tubes from freezer and centrifuge 30 min at 14,000 × g, 4°C.

  When GlycoBlue is used, you will be able to see a small blue pellet at the bottom of each 1.5‐ml tube (Fig. 5). Depending on the amount of ChIPed DNA, the small blue pellet may or may not be visible. If the pellet is not visible, it does not mean necessarily there is no DNA. In both cases, be extra careful when discarding the ethanol liquid.

Figure 5.

Image of ChIPed DNA pellets after precipitation and before resuspension (Basic Protocol 2, step 103). GlycoBlue is “optionally” used for a better visualization of the pellets (The pellets are indicated with the black arrows).

• 99Discard the liquid with extra care not to disturb the pellet.
• 100Add 700 µl of 70% ethanol.
• 101Centrifuge 3 min at 14,000 × g, 4°C.
• 102Discard the liquid with extra care, not to disturb the pellet.
• 103On a clean paper towel, put down the tubes with open lids and air‐dry the pellet at room temperature, until no wetness (no large drops of ethanol) is observed.
• 104Resuspend pellet in 10 µl nuclease‐free water. Mix by flicking the tube, then centrifuge as in step 32.

  Use 2 µl for Qubit dsDNA HS assay quantification, and store remaining 8 µl at –20°C until downstream applications (qPCR or library preparation for sequencing).

  Stopping point: Samples could be stored long‐term at –20°C.

  Avoid repetitive freeze‐thaw cycles, as it degrades sample and reduces the quality of downstream application output.

• 105Quantify the DNA from step 104 using Qubit dsDNA HS assay kit.

  The ChIPed DNA from this protocol may serve as suitable input for diverse downstream applications (i.e., qPCR, sequencing) and optimal for multiple library preparation strategies that are compatible with multiple downstream sequencing platforms.

Basic Protocol 3. LIBRARY CONSTRUCTION FOR DNA SEQUENCING

Here, we outline MGI library construction from the input DNA and ChIPed DNA samples from Basic Protocol 2, using MGIEasy UDB Universal Library Prep Set kit (MGI, cat. no. 1000022803). Libraries can be sequenced on Complete Genomics/MGI sequencing platform (e.g., DNBSEQ‐G99RS). Besides the low cost, several advantages of this alternative become apparent in the context of ChIP‐seq samples, including the use of PCR‐free DNBs amplification, which significantly reduces duplication rates and amplification bias. For most ChIP‐seq libraries, it is sufficient to sequence them at a depth of 30 to 50 million reads, which allows a library multiplexing of two samples per DNBSEQ‐G99 flow cell.

Materials

• ChIPed DNA and input DNA from Basic Protocol 2

• MGIEasy UDB Universal Library Prep Set kit (MGI, cat. no. 1000022803)

• 80% ethanol, freshly prepared

• H₂O, nuclease‐free (Ambion, cat. no. AM9937)

• Qubit dsDNA HS assay kit (Invitrogen, cat. no. Q32854)

• High Sensitivity D1000 kit (Agilent Technologies, cat. nos. 5067‐5584 and 5067‐5585)

• Qubit ssDNA assay kit (Invitrogen, cat. no. Q10212)

• 0.2‐ml individual or 8‐strip PCR tubes

• Ice bucket with ice

• Vortex mixer

• Mini centrifuge

• Thermocycler

• P‐200 pipette and tips

• 1.5‐ml DNA Lobind tubes

• Magnetic rack DynaMagTM‐2 (Thermo Fisher Scientific, cat. no. 12321D) or equivalent

• Agilent 2200 TapeStation (Agilent Technologies, cat. no. G2939AA) or equivalent

• Qubit assay tubes (Invitrogen, Q32856)

• Qubit 4 fluorometer (Thermo Fisher Scientific, cat. no. Q33216)

• 1Transfer up to 50 ng of samples (from Basic Protocol 2, step 104) to a new 0.2‐ml PCR tube (or 8‐strip, depending on the number of samples).
• 2Add TE buffer for a final volume of 39.5 µl per sample. Place the PCR tube(s) on ice.

• 3
  Prepare the ERAT mixture for (N + 1) number of samples for a total volume of 10.5 µl per sample as follows:

  • 6 µl ERAT Buffer
  • 4.5 µl ERAT Enzyme Mix.
• 4Transfer 10.5 µl ERAT mixture to each sample tube, vortex 3 times (3 s each), and centrifuge using the mini centrifuge 5 s at maximum speed, room temperature, to collect the solution at the bottom of the tube.
• 5
  Place the tube(s) into the thermocycler and run the following program with heated lid on (105°C):

  • 30 min at 20°C
  • 15 min at 65°C
  • Hold at 4°C.
• 6Briefly centrifuge as in step 4 to collect the solution at the bottom of the tube.

Adapter ligation

Dilute the adapters with TE buffer following manufacturer instructions. Sometimes the ChIPed DNA is unquantifiable. For starting DNA amounts <50 ng, we achieved successful results using a 10‐fold dilution.

• 7Add 5 µl of diluted adapter to each PCR tube from step 6. Pipette to mix (8 times) and briefly centrifuge as in step 4 to collect solution at the bottom of the tube(s).
• 8
  Prepare the adapter ligation mixture for (N + 1) number of samples for a total volume of 25 µl per sample as follows:

  • 21 µl Ligation Buffer
  • 4 µl DNA Ligase.
• 9Pipette slowly to transfer 25 µl of ligation mixture to the PCR tube from step 7.
• 10Vortex 6 times (3 s each) and briefly centrifuge as in step 4 to collect the solution at the bottom of the tube(s).
• 11
  Place the tube(s) into the thermocycler and run the following program with heated lid on (50°C):

  • 30 min at 23°C
  • Hold at 4°C.
• 12Centrifuge briefly as in step 4 to collect the solution at the bottom of the tube(s).
• 13Add 20 µl TE buffer for a total volume of 100 µl.
• 14Transfer all the solution to a new 1.5‐ml centrifuge tube.

  Stopping point: Adapter‐ligated DNA can be stored up to 16 hr at –20°C.

Cleanup of adapter‐ligated DNA

Allow DNA Clean Beads to come to room temperature for 30 min. Vortex and mix thoroughly before use.

• 15Transfer 50 µl DNA Clean Beads to the tube from step 14 and mix thoroughly by pipetting up and down (at least 10 times).
• 16Incubate at room temperature for 5 min.
• 17Briefly centrifuge as in step 4 and place the tube onto a magnetic separation rack for 2 to 5 min until the liquid becomes clear.
• 18Carefully remove and discard the supernatant with a P‐200 pipette.
• 19Keep the tube on the magnetic separation rack and add 200 µl of freshly prepared 80% ethanol to each tube.
• 20Incubate for 30 s, then carefully remove and discard the supernatant without disturbing the beads.
• 21Repeat steps 19 and 20 once.
• 22Keep the tube on the magnetic separation rack with the lid open, and air‐dry beads at room temperature until no wetness is observed, but before the pellet begins to crack.
• 23Remove the centrifuge tube from the magnetic separation rack and add 40 µl TE buffer to elute the DNA. Mix thoroughly by pipette up and down (at least 10 times).
• 24Incubate at room temperature for 5 min.
• 25Briefly centrifuge as in step 4 and place the tube back onto the magnetic separation rack for 2 to 5 min until the liquid becomes clear. Transfer 38 µl of supernatant to a new 0.2‐ml PCR tube(s).

  Stopping point: After cleanup, adapter‐ligated DNA can be stored up to 16 hr at –20°C.

• 26To each sample (from step 25) add 50 µl of PCR enzyme mix, then add 12 µl of a chosen UDB primer mix (barcode).

  It is recommended to follow manufacturer's instructions for barcode choice.

• 27Vortex 3 times (3 s each) and centrifuge briefly as in step 4 to collect the solution at the bottom of the tube(s).
• 28
  Place the tube into the thermocycler and run the following program with heated lid on (105°C).

  • Initial step: 3 min at 95°C
  • 14 cycles: 20 s at 98°C
  • 15 s at 60°C
  • 30 s at 72°C
  • Final step: 10 min at 72°C
  • Hold at 4°C.
• 29Centrifuge briefly as in step 4 to collect the solution at the bottom of the tube. Transfer all the solution to a new 1.5‐ml tube.

• 30Transfer 100 µl DNA Clean Beads to the tube from step 29 and mix thoroughly by pipetting up and down (at least 10 times).
• 31Incubate at room temperature for 5 min.
• 32Briefly centrifuge as in step 4 and place the tube onto a magnetic separation rack for 2 to 5 min until the liquid becomes clear.
• 33Carefully remove and discard the supernatant with a P‐200 pipette.
• 34Keep the tube on the magnetic separation rack and add 200 µl of freshly prepared 80% ethanol to each tube. Incubate for 30 s.
• 35Carefully remove and discard the supernatant without disturbing the beads.
• 36Repeat steps 34 and 35 once.
• 37Keep the tube on the magnetic separation rack with the lid open, and air‐dry beads at room temperature until no wetness is observed, but before the pellet begins to crack.
• 38Remove the centrifuge tube from the magnetic separation rack and add 32 µl TE buffer to elute the DNA. Mix thoroughly by pipette up and down (at least 10 times).
• 39Incubate at room temperature for 5 min.
• 40Briefly centrifuge as in step 4 and place the tube back onto the magnetic separation rack for 2 to 5 min until the liquid becomes clear. Transfer 30 µl of supernatant to a new 1.5‐ml tube.

  Stopping point: After cleanup, purified PCR Products can be stored at –20°C.

Quality control of PCR products

Use 1 µl of PCR product from step 40 to quantify the purified PCR products with dsDNA fluorescence assay kits such as the Qubit dsDNA HS Assay Kit. Assess the fragment size distribution of purified PCR products with Bioanalyzer or TapeStation (Agilent Technologies). Figure 6 shows the final size distribution ranging from 200 to 400 bp and a peak at ∼270 to ~300 bp.

Figure 6.

Assessment of library size distribution post‐amplification (Basic Protocol 3, step 40) using High Sensitivity D1000 ScreenTape kit, readout on Agilent 2200 TapeStation. (A) Gel image of all 4 libraries. L = ladder. (B) An electropherogram of sample T2 showing an optimal fragment distribution between 200 and 400 bp with a narrow peak at 270 bp.

Denaturation

Formulas 6 and 7 show the calculation of the mass (in ng) that corresponds to 1 pmol of a dsDNA sample with varying fragment sizes.

$$Mass\left(\mathrm{ng}\right)correspondingto1\mathrm{pmol}PCRproducts=\frac{\left[FragSize\left(\mathrm{bp}\right)\times 660\mathrm{ng}\right]}{1000\mathrm{bp}}$$ (6)

$$Volume\left(\mu \mathrm{l}\right)correspondingto1\mathrm{pmol}PCRproducts=\frac{Mass\left(\mathrm{ng}\right)}{Qconc\left(\mathrm{ng}/\mu \mathrm{l}\right)}$$ (7)

Where Frag Size = DNA fragment size from bioanalyzer/TapeStation and Qconc = concentration value from Qubit.

• 41Transfer the 1 pmol corresponding volume (from Formula 7) of PCR product to a new 0.2‐ml PCR tube. Add TE buffer to a final volume of 48 µl.
• 42Incubate the tube(s) into the thermocycler at 95°C for 3 min, with heated lid on (105°C).
• 43After the reaction is complete, immediately place the PCR tube on ice for 2 min, then centrifuge briefly as in step 4.

• 44
  Prepare the single strand circularization reaction mixture for (N + 1) number of samples, for a total volume of 12.1 µl per sample as follows:

  • 11.6 µl Splint Buffer
  • 0.5 µl DNA Rapid Ligase.
• 45Transfer 12.1 µl single strand circularization mixture to the PCR tube from step 43. Vortex 3 times (3 s each) and centrifuge briefly as in step 4 to collect the solution at the bottom of the tube.
• 46Incubate the tube(s) into the thermocycler at 37°C for 10 min, with heated lid on (105°C).
• 47After the reaction is complete, immediately place the tube on ice for the next reaction.

• 48
  Prepare the enzymatic digestion mixture for (N + 1) number of samples, for a total volume of 4 µl per sample as follows:

  • 1.4 µl Digestion Buffer
  • 2.6 µl Digestion Enzyme.
• 49Transfer 4 µl enzymatic digestion mixture into the PCR tube from step 47. Vortex 3 times (3 s each) and centrifuge briefly as in step 4 to collect the solution at the bottom of the tube.
• 50Incubate the tube(s) into the thermocycler at 37°C for 10 min, with heated lid on (105°C).
• 51Briefly centrifuge as in step 4 to collect the solution at the bottom of the tube.
• 52Add 7.5 µl Digestion Stop Buffer to the PCR tube. Vortex 3 times (3 s each) and centrifuge briefly to collect the solution at the bottom of the tube. Transfer all the solution into new 1.5‐ml tube.

• 53Transfer 170 µl of DNA Clean Beads to the enzymatic digestion product from step 52. Gently pipette at least 10 times to mix thoroughly.
• 54Incubate at room temperature for 10 min.
• 55Centrifuge briefly as in step 4 and place the 1.5‐ml tube on the magnetic rack for 2 to 5 min until the liquid becomes clear. Carefully remove and discard the supernatant using a pipette.
• 56With the 1.5‐ml tube on the magnetic rack, add 500 µl of freshly prepared 80% ethanol to the tube without disturbing the beads. Incubate for 30 s. Carefully remove and discard the supernatant.
• 57Repeat step 56 once.
• 58Keep the 1.5‐ml centrifuge tube on the magnetic rack with the lid open, and air‐dry beads until no wetness is observed, but before the pellet cracks.
• 59Remove the 1.5‐ml centrifuge tube from the magnetic rack and add 22 µl TE Buffer to elute the DNA. Gently pipette up and down at least 10 times to mix thoroughly or until the beads are fully resuspended.
• 60Incubate at room temperature for 10 min.
• 61Centrifuge briefly as in step 4 and place the 1.5‐ml centrifuge tube back on the magnetic rack for 2 to 5 min until the liquid becomes clear. Transfer 20 µl of supernatant to a new 1.5‐ml tube.

  Stopping point: After cleanup, purified products can be stored at –20°C. The final product of this process is single stranded Circular DNA (ssCirDNA).

• 62Quantify the purified enzymatic digestion products with Qubit ssDNA Assay Kit. This quantification is required for downstream calculations in DNBs preparation.

Basic Protocol 4. DNA NANOBALLS PREPARATION FOR THE DNBSEQ‐G99RS SEQUENCING PLATFORM AND DATA QUALITY CONTROL

In Complete Genomics/MGI‐DNBSEQ technology, DNBs preparation is an equivalent of cluster generation in other short‐read sequencing platforms, with major differences being that it is a PCR‐free process and that it occurs off the flow cell (in tube). The process of making DNBs is based on the amplification of the ssCirc DNA (from Basic Protocol 3, step 60) using the rolling circle replication (RCR) method (Drmanac et al., 2010).

Materials

• DNA libraries from Basic Protocol 3

• DNBSEQ‐G99RS high‐throughput sequencing reagent set V2.0 (Complete Genomics, cat. no. 940‐001871‐00)

• Qubit ssDNA assay kit (Invitrogen, cat. no. Q10212)

• Vortex mixer

• Mini centrifuge

• Ice bucket with ice

• Thermocycler

• Wide‐bore, non‐filtered pipette tip and pipette

• Qubit assay tubes (Invitrogen, Q32856)

• Qubit 4 fluorometer (Thermo Fisher Scientific, cat. no. Q33216)

• Complete Genomics/MGI sequencing platform (e.g., DNBSEQ‐G99RS)

Library input requirement

For libraries that are constructed using MGIEasy UDB Universal Library Prep Set kit (MGI, cat. no. 1000022803) that involve PCR amplification step, the required concentration of circular ssDNA library input should be ≥2 fmol/µl. The following formula is used to convert the concentration of the ssDNA library from ng/µl to fmol /µl.

$$Conc\left(\mathrm{fmol}/\mu \mathrm{l}\right)=\frac{\left[3030\times Qconc\left(\mathrm{ng}/\mu \mathrm{l}\right)\right]}{FragSize\left(\mathrm{bp}\right)}$$ (8)

Where Frag Size = fragment size from bioanalyzer/TapeStation analysis (quality control of PCR products step; Basic Protocol 3) and QConc = concentration value from Qubit (ssDNA) from step 62 (Basic Protocol 3).

Making DNBs

The required volume of ssDNA libraries is 20 fmol per library. The corresponding volume to 20 fmol is calculated using the following formula.

$$Volume20\mathrm{fmol}\left(\mu \mathrm{l}\right)=\frac{20\mathrm{fmol}}{Conc\left(\mathrm{fmol}/\mu \mathrm{l}\right)}$$ (9)

Where Volume 20 fmol (µl) = volume in µl corresponding to 20 fmol of each library product and Conc (fmol/µl) = concentration value from formula 8.

At this stage, we pool two ChIP libraries (ssDNA#1 and ssDNA#2) to make a single DNB reaction mix as follows.

$$V_L\left(\mu \mathrm{l}\right)=Volume\left(20\mathrm{fmol}\right)ofssDNA\#1+Volume\left(20\mathrm{fmol}\right)ofssDNA\#2$$ (10)

Where V_L = volume of pooled library.

• 1
  Prepare the Make DNB reaction mixture 1 as follows:

  • V_L (volume of pooled library, i.e., ssDNA#1 + ssDNA#2)
  • 10 µl – V_L (volume of low TE buffer)
  • 10 µl Make DNB buffer.
• 2Mix the reaction mixture thoroughly by using a vortex mixer. Centrifuge using the mini centrifuge 5 s at maximum speed, room temperature, and place it on ice until use.
• 3
  Place the tube into the thermocycler and run the following program with heated lid on (105°C):

  • 1 min at 95°C
  • 1 min at 65°C
  • 1 min at 40°C
  • Hold at 4°C.
• 4Remove the PCR tube from the thermocycler when the temperature reaches 4°C.
• 5
  Centrifuge using the mini centrifuge 5 s at maximum speed, room temperature, place the tube on ice, and prepare Make DNB reaction mixture 2 as follows:

  • 20 µl Make DNB Enzyme Mix I
  • 2 µl Make DNB Enzyme Mix II (LC).
• 6Add all the Make DNB reaction mixture 2 into Make DNB reaction mixture 1 from step 4. Mix thoroughly by using a vortex mixer. Centrifuge as in step 5.
• 7Place the tube into the thermocycler and incubate at 30°C for 20 min with heated lid on (35°C) and hold at 4°C.
• 8Immediately add 10 µl of Stop DNB Reaction Buffer when the temperature reaches 4°C and place the reaction mixture tubes on ice. Mix gently by pipetting 8 times using a wide‐bore, non‐filtered pipette tip.

Quantifying and pooling DNBs

When the making DNB process is completed, take 2 µl of DNBs, and use the Qubit ssDNA Assay Kit and Qubit fluorometer to quantify the DNBs. An ideal concentration of ∼8 ng/µl would be recommended, although we had successful sequencing runs with less than that.

DNBSEQ‐G99RS high‐throughput sequencing

The prepared DNBs are sequenced using the Complete Genomics/MGI‐G99 sequencer. DNBs are loaded onto a flow cell with a patterned array for optical sequencing. Average data output will vary with different library types and applications. When using App‐D FCL PE150 of DNBSEQ‐G99RS High‐throughput Sequencing Reagent Set V2.0 (Complete Genomic, cat. no. 940‐001871‐00), the average data output is ∼24 Gb (80 to 120 million reads), which allows running two ChIP libraries per flow cell for 30 to 50 million reads per sample. The run lasts ∼12 hr, and at the end, the machine generates a summary report with quality control metrics including Q30, estimated error rate, barcode split rate, base distribution, and read quality (see Supporting Information, Figs. S2 to S7).

REAGENTS AND SOLUTIONS

Cross‐linking solution, 1%

For each sample, prepare 30 ml of a 1% cross‐linking solution by diluting 811 µl formaldehyde (37% formaldehyde stock solution, Fisher BioReagents, cat. no. BPBP531500) in 29.189 ml of cold 1× PBS (Current Protocols). Prepare fresh.

D₀‐buffer: Dilution buffer

• 20 mM Tris·HCl, pH 8.0 (Current Protocols, 2006)

• 1 mM EDTA, pH 8.0 (Current Protocols, 2006)

• 1% Triton X‐100 (Current Protocols, 2006)

• 0.1% Na‐deoxycholate (Fisher Scientific, cat. no. 50‐255‐883)

• Bring volume to 48 ml with sterile Milli‐Q water

• Store up to 1 month at 4°C

  D₁‐buffer: Just before use, aliquot the required amount of D₀‐buffer and supplement it with 150 mM NaCl (Current Protocols, 2006). Place on ice.

  D₂‐buffer: Just before use, aliquot required amount of D₀‐buffer and supplement it with 150 mM NaCl (Current Protocols, 2006) and with 1× protease inhibitors (Fisher Scientific, cat. no. PIA32963). Place on ice.

E‐buffer: Elution buffer

• 50 mM Tris·HCl, pH 8.0 (Current Protocols, 2006)

• 1 mM EDTA, pH 8.0 (Current Protocols, 2006)

• 1% sodium dodecyl sulfate (SDS) (Current Protocols, 2006)

• 50 mM sodium bicarbonate (NaHCO₃) (Current Protocols, 2006)

• Bring volume to 50 ml with sterile Milli‐Q water

• Store up to 6 months at room temperature

Glycine solution, 2.5 M

• 93.8 g glycine (Fisher Scientific, cat. no. BP381‐5)

• 500 ml dH2O

• Filter using a 0.22‐µm filter

• Store up to 6 months at room temperature

L₀‐buffer: Lysis buffer

• 25 mM of HEPES (Current Protocols, 2006)

• 10 mM KCl (Current Protocols, 2006)

• 1.5 mM MgCl₂ (Current Protocols, 2006)

• 0.1% NP‐40 (Millipore, cat. no. 492018)

• Bring volume to 49.5 ml with sterile Milli‐Q water

• Store up to 1 month at 4°C

  L₁‐buffer: Just before use, aliquot required amount of L₀‐buffer and supplement it with 1× protease inhibitors (Fisher Scientific, cat. no. PIA32963). Place on ice.

PBS supplemented with protease inhibitors

Just before use, aliquot required amount of 1× PBS (Current Protocols, 2006) and supplement it with 1× protease inhibitors (Fisher Scientific, cat. no. PIA32963). Prepare fresh and place on ice.

PBS supplemented with Tween 20 (PBST)

• 1× PBS (Current Protocols, 2006)

• 0.1% Tween 20 (Current Protocols, 2006)

• Store up to 1 month at 4°C

S₀‐buffer: Sonication buffer

• 10 mM Tris‐HCl, pH 7.6 (Current Protocols, 2006)

• 1 mM EDTA, pH 8.0 (Current Protocols, 2006)

• 0.1% SDS (Current Protocols, 2006)

• Bring volume to 49.5 ml with sterile Milli‐Q water

• Store up to 1 month at 4°C

  S₁‐buffer: Just before use, aliquot required amount of S₀‐buffer and supplement it with 1× protease inhibitors (Fisher Scientific, cat. no. PIA32963). Place on ice.

WA₀‐buffer: Wash buffer A

• 50 mM Tris·HCl, pH 8.0 (Current Protocols, 2006)

• 1 mM EDTA, pH 8.0 (Current Protocols, 2006)

• 1% Triton X‐100 (Current Protocols, 2006)

• 0.1% Na‐deoxycholate (Fisher Scientific, cat. no. 50‐255‐883)

• 0.1% SDS (Current Protocols, 2006)

• Bring volume to 44.5 ml with sterile Milli‐Q water

• Store up to 1 month at 4°C

  WA₁‐buffer: Just before use, aliquot required amount of WA₀‐buffer and supplement it with 500 mM NaCl (Current Protocols, 2006) and 1× protease inhibitors (Fisher Scientific, cat. no. PIA32963). Place on ice.

WB₀‐buffer: Wash buffer B

• 20 mM Tris·HCl, pH 8.0 (Current Protocols, 2006)

• 1 mM EDTA, pH 8.0 (Current Protocols, 2006)

• 250 mM LiCl (Millipore, cat. no. 5922)

• 0.5% NP‐40 (Millipore, cat. no. 492018)

• 0.5% Na‐deoxycholate (Fisher Scientific, cat. no. 50‐255‐883)

• Bring volume to 49.5 ml with sterile Milli‐Q water

• Store up to 1 month at 4°C

  WB₁‐buffer: Just before use, aliquot required amount of WB₀‐buffer and supplement it with 1× protease inhibitors (Fisher Scientific, cat. no. PIA32963). Place on ice.

COMMENTARY

Background Information

Understanding tissue‐specific changes in protein–DNA interactions helps reveal how gene expression is regulated in major biological processes, such as development, aging, and cancer (Emig & Albrecht, 2011; Ferraz et al., 2021; Hall, 2011); the development of techniques such as ChIP has contributed significantly to our understanding of protein–DNA interactions in vivo (Agbleke et al., 2020; Chen et al., 2024). As genome‐wide technologies advanced, ChIP was combined with next‐generation sequencing (ChIP‐seq), allowing high‐resolution mapping of protein‐DNA interactions. Thus, ChIP‐seq has become a gold standard method in genomic and epigenomic research, and has been used to answer a wide array of biological questions, including mapping transcription factor binding sites, profiling histone modifications, studying chromatin organization, and exploring these processes in both health and disease across a variety of organisms (Gade & Kalvakolanu, 2012; Kidder et al., 2011; Mundade et al., 2014; Northrup & Zhao, 2011). The small number of ChIP‐seq protocols specifically developed for tissue samples comes with its own limitations, such as tissue‐type specificity, labor‐intensive procedures, complex optimization, or the need for costly equipment and reagents.

In this article, we propose a refined series of methods optimized to address several key limitations discussed above. Certain tissues are naturally difficult to homogenize due to their structural characteristics and heterogeneous composition. This challenge is further compounded by the presence of fats and connective fibers, which make the tissues slippery and resistant to disruption (Graham, 2002). The protocol offers the flexibility to select either a Dounce grinder or the gentleMACS Dissociator for homogenization, depending on availability and specific experimental requirements. However, it is important to optimize the homogenization conditions for each method to ensure efficient tissue disruption. We introduced extra wash steps with lysis and sonication buffers to reduce cytoplasmic and non‐specific background and increase recovery of intact nuclei. We optimized the buffer composition to ensure effective shearing of both tissue samples and cultured cells, obtaining consistent results across various tissues and cell types. Any sonicator can be used for chromatin shearing if proper optimization of run conditions is performed on test samples prior to run study samples. The subsequent steps of immunoprecipitation and reverse cross‐linking are optimized to enhance the quality of ChIPed DNA, with multiple wash steps to reduce the background noise. One key advantage of our protocol is that the ChIP‐enriched DNA may serve as a suitable input for diverse downstream applications (i.e., qPCR, sequencing) and is optimal for multiple library preparation strategies that are compatible with multiple downstream sequencing platforms.

Critical Parameters

Controls

It is important to use appropriate controls to ensure accuracy and consistency of ChIP‐seq experiments and increase reliability of the biological insights while minimizing artifacts. The types of controls most frequently used are input DNA, IgG controls, and spike‐in controls, each serving a specific purpose in validating and interpreting the data. Input DNA is one of the most used controls and represents a sample of sheared chromatin that has not undergone immunoprecipitation and helps account for biases introduced during chromatin shearing, DNA purification, and sequencing. In Basic Protocols 1 and 2, we described the steps of input control preparation. Whole genome sequencing data from the same patients were made available as part of a large cohort study, which eliminated our need for separately sequencing input controls (Terry Fox Research Institute Marathon of Hope Cancer Centers Network. Electronic address: mmarra@bcgsc.ca & Terry Fox Research Institute Marathon of Hope Cancer Centers Network, 2025). IgG controls use a non‐specific antibody to detect background signals resulting from non‐specific binding to beads or chromatin, helping to identify noise unrelated to the target protein. However, in cases where the chromatin amount is a limiting factor, i.e., in human tissue biopsies, IgG controls are practically unconceivable. Moreover, IgG antibodies are sought to typically yield significantly less DNA than specific antibodies, which can lead to over‐amplification of a limited set of genomic regions during library preparation, resulting in inadequate genome‐wide coverage for use as an effective background model in peak identification. For spike‐in normalization, the use of spike‐in controls is often impractical due to the heterogeneous nature of tumor tissues (Kidder et al., 2011; Patel et al., 2024).

Processing temperatures

An important critical factor to consider for a successful ChIP assay is the handling and processing of samples under consistently cold conditions. Inappropriate handling of frozen tissues, such as handling at room temperature or warming by hand, can lead to rapid enzymatic degradation of proteins and the loss of DNA–protein interactions. Apart from cross‐linking and quenching with glycine steps, all steps involved in Basic Protocols 1 and 2, including tissue preparation, lysis, chromatin isolation and shearing, immunoprecipitation, and washes, should be carried out on ice using ice‐cold buffers, freshly supplemented with protease inhibitors (as indicated). This is the case up until the elution step, where chromatin–protein complexes are released from the beads and temperatures may be briefly raised under controlled conditions for downstream applications, such as reverse cross‐linking and DNA extraction.

Homogenization

A first step of mincing is crucial to allow efficient homogenization by cutting through resistant connective fibers and fatty tissues. Due to the small size of the tissue samples, mincing with a blade is preferred over mortar grinding because it is faster and easier, and it reduces contamination risks and loss of material adhering to the mortar surface. Due to size variability, optimization of the number of strokes for the Dounce grinder, and the run program for gentleMACS Dissociator, should be performed prior to running study samples. The gentleMACS machines have a selection of pre‐defined programs optimized for the most frequently used tissues. While vortexing the samples using the gentleMACS Dissociator, ensure that the sample is in direct contact with the blade rather than settling at the bottom of the C‐tube. Otherwise, proper homogenization will not occur. Note that PBS will turn pink as an indication of good homogenization.

Cross‐linking

The efficiency of cross‐linking can be influenced by factors, such as the concentration of formaldehyde and the presence of glycine, the quenching agent. Formaldehyde can capture both DNA–protein and protein–protein interactions. Depending on the study's purpose and the molecular interactions involved, it is crucial to optimize both the formaldehyde concentration and the incubation time (Hoffman et al., 2015). A concentration of 1% cross‐linking solution for 10 min is sufficient to ensure efficient cross‐linking of DNA–protein complexes while minimizing the artifactual capture of non‐specific interactions. Reverse cross‐linking at 65°C for 4 hr in the presence of 200 mM NaCl enhances reversal efficiency.

Chromatin shearing

A key step to a successful ChIP‐seq assay is the efficient chromatin shearing. It is important to achieve consistent shearing results before proceeding with study samples. Whether using the Diagenode Pico Bioruptor or another sonication device, make sure to perform optimization first. Depending on the tissue or cell type, parameters of power, number of cycles, on/off times need systematic optimization. If you are using a probe sonicator, take into consideration risks, such as cross‐contamination, inconsistent shearing period and power, deficient cooling system (ice bucket in general), and the age and condition of the probe. The chromatin shearing process should be performed at very low temperatures to preserve sample integrity. An optimal range of fragment size is between 150 and 700 bp.

Magnetic beads

We recommend the use of magnetic beads for their clean, rapid, and straightforward isolation of complexes using magnetic racks, reducing the multiple centrifugations during wash steps, which saves time. We use Protein A‐conjugated Dynabeads for their compatibility with a wide range of antibodies and enhancement of the ChIP signal with lower amount of antibody used (1 µg). During wash steps involving beads bound to antibody‐chromatin complexes, the 5 min rotation at 4°C was optimized to ensure gentle yet complete resuspension of the beads to prevent disruption of complexes.

ChIP‐grade antibody

Another key factor to the success of a ChIP‐seq assay is the antibody quality, its high specificity, and high affinity to the protein of interest. Many antibodies commercialized as “ChIP‐grade” are not necessarily effective in ChIP assays. For that, a test of the antibody should be performed to validate its efficiency for a ChIP experiment. Antibody cross‐reactivity with protein of interest should be evaluated by western blotting prior to ChIP experiments. It is recommended to include appropriate controls to distinguish true binding events from background noise (Kidder et al., 2011).

MGI library construction

In Basic Protocol 3, it is crucial to follow the manufacturer's instructions meticulously to ensure successful library preparation, particularly during pipetting steps, centrifugation, vortexing periods, and the selection of UDB barcodes. Only freeze samples at the recommended stopping points. Additionally, the quality and quantity of adapters directly affect the efficiency and quality of library construction, and it is recommended to follow manufacturer's instructions for dilution rates according to input amount. Another critical parameter is the number of PCR cycles. Fewer cycles may result in a lower library yield, while more cycles can lead to increased PCR duplicates, chimeric sequences, or accumulated mutations.

Troubleshooting

In the previous section, we outlined the critical parameters to consider when performing this protocol for a successful ChIP‐seq assay, including some recommendations that will help troubleshooting potential challenges that might be encountered during the procedure. Table 1 highlights some additional challenges.

Table 1.

Troubleshooting Guide for ChIP Experiment

Problem	Possible cause	Solution
Tiny or invisible nuclei pellet	Deficient cell lysis	Step 12 (Basic Protocol 2) is a quick wash step prior to lysis; avoid leaving the cells in the lysis buffer for an extended period, and proceed quickly to centrifugation
		In step 16 (Basic Protocol 2), ensure proper rotation and thorough resuspension of the cells in the lysis buffer
Inconsistent size distribution of chromatin fragments	Poor chromatin shearing	Ensure proper operation of sonication device
		Optimize the number of cycles and the shearing periods (ON/OFF times)
		Check optimal volume of sample suspension for proper sonication
Low yield of ChIPed DNA	Antibody quality	Use ChIP‐grade antibodies and confirm that the target epitope is accessible
	Over/under cross‐linking	Avoid delays after adding cross‐linking solution to the sample and before quenching with glycine
	Expired reagents	Use unexpired reagents
	Disruptive washes	Ensure gentle addition of buffer during wash steps to avoid DNA–protein complex dissociation
No enrichment	Antibody quality	Use ChIP‐grade antibodies and confirm epitope targeting
Lower library yield	Insufficient cycles of amplification	The number of PCR cycles must be strictly controlled

Understanding Results

This article outlines a series of Basic Protocols that allow successful and reproducible ChIP‐seq experiments from cells or tissues, enabling the study of specific protein–chromatin interactions or insights into chromatin states through histone mark analysis. A successful result for these protocols is defined by a high‐quality sequencing run (as shown in Supporting Information, Figs. S2 to S7). Whether running the ChIPed samples on the MGI platform or other next‐generation sequencing platform, the choice of library preparation kit and sequencing platform should not affect the result.

We present here an example of information obtained from a bioinformatic analysis of a ChIP‐seq experiment targeting H3K27ac marks in four tissue samples: two tumors (T1 and T2) and their adjacent normal tissues (NT1 and NT2) as shown in Supporting Information, Figure S1. Pair 1 (T1 and NT1) was homogenized using genleMACS Dissociator, while pair 2 (T2 and NT2) was homogenized manually with Dounce grinder. ChIP‐seq FASTQ files were aligned to the GRCh38 (hg38) human reference genome using BWA‐MEM v0.7.17‐r1188 (Li, 2013). The reference included all alternate contigs, decoy sequences, and unplaced scaffolds to ensure accurate read mapping. Quality control was performed using FastQC v0.11.9 and SAMStat v2.2.3 (Lassmann, 2023). Sequencing quality and alignment metrics indicated that all four ChIP‐seq datasets were of high quality (see Supporting Information). The number of sequenced reads ranged from 50 to 60 million per sample, with >97% of reads properly paired across all datasets. Most aligned reads had high mapping quality (MAPQ >30), with values exceeding 90% across all samples, indicating confident and specific alignments to the reference genome. Reads with low mapping quality (MAPQ <10) remained below 8% for all samples, and unmapped reads were consistently under 1.5%, suggesting minimal contamination or adapter artifacts (Table 2). These results confirm the reliability of the data for downstream ChIP‐seq analysis. As an example of biologically relevant signal enrichment, we visualized the TP53 locus using IGV (Robinson et al., 2011). In the context of colorectal cancer, the TP53 promoter is expected to be actively marked by H3K27ac, reflecting transcriptional activity of this key tumor suppressor. As shown in Figure 7, all four samples demonstrated clear H3K27ac enrichment at the TP53 promoter, supporting both the antibody specificity and the quality of the ChIP‐seq libraries.

Table 2.

ChIP‐seq Quality Control Summary

Sample	Reads (million)	Properly paired (%)	MAPQ >30 (%)	MAPQ <30 (%)	MAPQ <10 (%)	Unmapped (%)
A1	103	98.8	92.40	0.56	6.75	0.29
B1	124	98.5	91.06	0.78	7.74	0.38
C1	103	98.6	91.32	0.77	7.24	0.67
D1	123	97.7	90.27	0.73	7.62	1.38

Figure 7.

H3K27ac ChIP‐seq signal at the TP53 locus in colorectal cancer samples. Integrated Genomics Viewer (IGV) screenshot showing aligned H3K27ac ChIP‐seq reads across the TP53 locus on chromosome 17p13.1. Each track represents one of the four samples. For each sample, the gray coverage histogram indicates the read depth across the region, highlighting peaks of enrichment at the TP53 promoter. Individually aligned reads are shown below the coverage track in purple, with colored bars indicating sequence mismatches or indels relative to the reference genome. The dense accumulation of reads upstream of the TP53 transcription start site reflects strong H3K27ac enrichment, consistent with active chromatin. The visualization confirms robust and consistent signal at a known active regulatory region across all samples, supporting both the specificity of the antibody and the technical quality of the ChIP‐seq experiment.

Time Considerations

Tissue preparation duration depends on the number of samples to be processed. This protocol was optimized for four samples (two tumors and two adjacent normal tissues) to process them as quickly as possible prior to cross‐linking. The mincing and homogenization procedures for these four samples take ∼20 min. Basic Protocol 2 begins with formaldehyde cross‐linking and quenching with glycine, which together require ∼30 min of hands‐on time. This is followed by four major chronological steps: lysis/sonication, immunoprecipitation, washes/elution, and DNA purification. Depending on the optimization of number of cycles and the on/off time settings of the sonication device, the entire lysis/sonication steps take 80 to 100 min to yield sheared chromatin. After this, it is recommended to store extracted chromatin at 4°C, while performing reverse cross‐linking of a 25‐µl aliquot at 65°C overnight for quality check. On the following day, after confirming the chromatin quality and ensuring samples are suitable for downstream steps, immunoprecipitation begins with bead preparation (∼3 hr), followed by incubation of the samples with the bead–antibody mixture at 4°C overnight. The third phase begins the next day with successive washes (60 to 80 min) and an elution step (∼30 min). The final step, DNA purification, includes heat incubation at 65°C for 4 hr. DNA precipitation for 1 hr at –80°C is faster, useful when time is a limitation factor, and results in a good yield, although it may coprecipitate other components such as salts. For maximum purity, however, it is recommended to precipitate DNA at –20°C overnight. For Basic Protocol 3, library construction using the MGI kit takes 1 to 1.5 days: ∼55 min for ERAT; ∼50 min for adapter ligation; 30 to 50 min for the first cleanup; 50 to 60 min for PCR amplification; 30 to 50 min for the second cleanup; 50 to 80 min for denaturation, circularization, and enzymatic digestion; and 50 to 60 min for the last cleanup. Basic Protocol 4 involves DNBs preparation, which takes 50 to 60 min. Cartridge preparation, priming, and flow cell loading require an additional 60 to 80 min. The sequencing run itself takes ∼12 hr (Fig. 8).

Figure 8.

Comprehensive timeline of the ChIP‐seq workflow across Basic Protocols 1‐4.

Author Contributions

Hayley Alloway: Investigation; methodology; writing—review and editing. Louisa Wiede: Resources. Daniel Loos: Resources. John Thoms: Resources. Khadija Rebbani: Conceptualization; methodology; writing—original draft. Touati Benoukraf: Conceptualization; formal analysis; funding acquisition; methodology; supervision; writing—review and editing.

Conflict of Interest

The authors declare no conflict of interest.

Supporting information

Figure S1:Variability in tissue sample sizes processed using the Refined ChIP‐seq protocol. Figures S2‐S7: in‐built quality assessment of a run in DNBseq‐G99RS.

FASTQC quality assessment of raw ChIP‐seq data generated for 4 tissue samples.

Alloway, H. , Wiede, L. , Loos, D. , Thoms, J. , Rebbani, K. , & Benoukraf, T. (2025). Refined chip‐seq protocol for high‐quality chromatin profiling in solid tissues using the complete genomics/MGI sequencing platform. Current Protocols, 5, e70260. doi: 10.1002/cpz1.70260

Data Availability Statement

The data that support the protocol were stated in the manuscript. Raw sequencing data of this protocol are available from the corresponding author (T.B.) upon reasonable request as they are part of a large cohort study by Marathon of Hope Cancer Centres Network (MOHCCN).