Purification of immune-active macrophage super enhancers by chemical cross-linked chromatin immune precipitation

Summary

Isolation of extraordinarily long-length super-enhancers (SEs) using typical chromatin immune precipitation (ChIP) techniques can lead to DNA breakage due to uncontrolled cross-linking. We present a redefined ChIP technique for SE purification. After controlled paraformaldehyde-based cross-linking, glycine was used to quench the cross-linker followed by mild sonication. The sonication produced ideal fragment length of long-length SE chromatin. Presently, miR146a-5p SE of macrophages was pulled using BRD4 protein. Our protocol can reproducibly simplify the SE element isolation issues, in a quality-controlled manner.

For complete details on the use and execution of this protocol, please refer to Das et al. (2021).¹

Graphical abstract

Highlights

• •miRNA SE control over a long chromatin distance, thus gaining therapeutic interest
• •Purification of large mediator complex occupied SE by standard ChIP problematic
• •Controlled chemical cross-linking and mild sonication can purify SE elements
• •Our protocol is validated in macrophages, cancer cell lines, and mice T cells

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Isolation of extraordinarily long-length super-enhancers (SEs) using typical chromatin immune precipitation (ChIP) techniques can lead to DNA breakage due to uncontrolled cross-linking. We present a redefined ChIP technique for SE purification. After controlled paraformaldehyde-based cross-linking, glycine was used to quench the cross-linker followed by mild sonication. The sonication produced ideal fragment length of long-length SE chromatin. Presently, miR146a-5p SE of macrophages was pulled using BRD4 protein. Our protocol can reproducibly simplify the SE element isolation issues, in a quality-controlled manner.

Before you begin

Endotoxin stimulation induces formation of extraordinary cis-acting elements of DNA known as Super-Enhancers (SEs) in order to induce persistent transcription of some immune regulatory miRNAs. SEs are longer than normal enhancers (>25 kB), occupied by unique histone modifiers and co-activator proteins like Bromo Domain protein 4, p300 histone acetyl transferase (p300 HAT) and RNA polymerase II (Rbp I). p300 HAT maintains H3K27 acetylation mark throughout such active SEs regions.² These SEs confer significant fine tuning on immune regulatory miRNAs over a very long distance (> 1 mbs). Therefore, such SEs can be a potential therapeutic target of interest for immune-suppressive diseases like visceral leishmaniasis, cancer etc. Unlike normal enhancers, available techniques like standard chromatin immune-precipitation (ChIP) protocols are quite inefficient in pulling down such a long DNA like SE element. The major issues occur mainly during the cross-linking and sonication steps. Inappropriate cross-linking and uncontrolled sonication can lead into loosing large mediator protein complexes from SE regions and eventually shearing of long SE elements.

This current protocol is a modified version of the available ChIP protocol mainly at cross-linking and sonication steps in order to make it suitable for immune-precipitation of active SEs from mouse bone marrow derived macrophages (mBMDMs) that are either stimulated or infected with Leishmania donovani parasites (Table 1). Besides mBMDMs, we have successfully implicated the protocol in RAW 264.7 macrophage cell lines and cancer cell stimulated T cells as well. Before beginning the steps of X-ChIP, following parameters are needed to be ensured.

• 1.Stimulate and activate a sufficient number of cells (20–30 × 10⁷ BMDMs/femur) before beginning.
• 2.Keep all the eppendorfs (both 1.5 mL and 2 mL), scraper and plasticwares autoclaved and sterile. Add complete protease inhibitor cocktail to the buffers on the same day at least ∼20 min prior beginning the work.
• 3.Be prepared with adequate quantities of buffer for both pilot- and batch-experiments.
• 4.Perform the cross-linking step by freshly prepared (< 1 week old) stock of 4% paraformaldehyde in 20 mM PBS (pH-7.5).

Note: In general, mBMDMs are reportedly subjected to cross-linking using 1% paraformaldehyde for 15 min followed by quenching with 125 mM glycine. In case of conventional ChIP, 35 s pulses followed by 45 s rest is given for 10 cycles in order to obtain <500 bp fragments of chromatin regions.³

Table 1.

Comparative parameters between conventional ChIP and X-ChIP

Parameters	Conventional ChIP	X-ChIP
Cross-linker percentage	2% Formaldehyde from 37% commercial stock solution	0.75% Paraformaldehyde in 20 mM PBS (pH-7.5)
Cross-linking timing	5 min at 30°C	10–15 min at 37°C
Quencher	0.75–1.5 M Tris (pH-4.5)	125 mM Glycine
Sonication	8-10 sonication cycles of 15″ on and 30″ off pulse	10 sonication cycles of 15″ on and 45″ off pulse

In our case the percentage of formaldehyde, sonication pulse and cycle has been modified to obtain controlled fragmentation and fragment length >500 bp.

Institutional permissions

Animal ethical clearance for BALB/c mice- A prior permission from institutional animal ethical committee should be obtained for raring, maintenance and euthanatizing of animals (here it is BALB/c mice).

Note: We have prioritized the age group of animals while choosing the litter mates and mostly kept it within 4–6 weeks because young animals contain sufficient number of healthy bone marrow derived monocytes that can be easily differentiated into macrophages.

Cell culture

Timing: 4–7 days

• 5.
  Parasite passaging to obtain infective stationary phase promastigotes.

  • a.
    On Day 0, transform 1–2 × 10⁶/mL L. donovani promastigotes by passaging in M199 media supplemented with 10% FBS.
  • b.
    Keep the culture in 22°C incubator under gentle shaking condition with 1,000 × g speed for seven days. On Day 7, check whether stationary phase metacyclic parasites are formed under 10× and 40× objective of light microscopy (Figure 1, Methods video S1). These parasites are used for infecting macrophages (Methods video S2).
• 6.
  Macrophage culture maintenance.

  • a.
    Isolation of mBMDMs from BALB/c mice.

    • i.
      4–6 weeks old BALB/c mice are euthanatized by cervical dislocation and the femur bones are collected for bone marrow isolation using 21G Dispovan ® syringe needle in sterile condition. Culture the bone marrow cells in Dulbecco’s modified eagles’ media supplemented with 10 ng/mL M-CSF for 6 days.
    • ii.
      On Day 6, replace the M-CSF containing media with normal DMEM and culture for another one day before stimulation or infection (Figure 1).
• 7.
  Macrophage stimulant preparation.

  • a.
    Stimulate or inhibite the formation of SEs in macrophage by the following steps:

    • i.
      For active SE formation, give chronic endotoxin shock with lipopolysaccharide (LPS).
    • ii.
      For inhibiting active SE formation, use JQ-1, a known inhibitor of the BET family of bromodomains.

Figure 1.

Flow chart of differentiation and stimulation of bone marrow derived macrophages by lipopolysaccharide or JQ1 and infection with L. donovani parasites

Outline of BMDM differentiation and stimulation and / or infection protocol has been provided in Figure 1.

Primer designing

Timing: 45 min

• 8.
  Primers were designed to amplify the small sections of BRD4 occupied SEs as follows:

  • a.
    Previously published two sets of ChIP-Seq data showed BRD4 enrichment at upstream cis-acting element of second exon of miR146a-5p encoding gene at mouse chromosome (LOC285628).^4,5 The primer flanking the super enhancer element of miR146a-5p has been obtained from this data repository.
  • b.
    The primers of 17–18 nucleotide length are procured from GCC Biotechnology, India. 100 μM primer stock, prepared by dissolving the lyophilized primer in nuclease-free water, can be stored at −80°C for 4–6 months.
  • c.
    Prepare working stock of 25 μM by diluting the main stock 4-times. It can be store at −20°C for 4–6 months.

CRITICAL: Primers should be vortexed properly and water should be completely free from nuclease. Avoid primer freeze thawing to prevent damage and degradation of primers.

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
Brd4	Cell Signaling Technology	BRD4 (E2A7X) Rabbit mAb
Chemicals, peptides, and recombinant proteins
M199	Sigma-Aldrich	56312C
DMEM high glucose	Gibco-Thermo Scientific	11965092
Fetal bovine serum, heat inactivated, Australia	Gibco-Thermo Scientific	10100147
Paraformaldehyde	Sigma-Aldrich	#158127
RNase A	Qiagen	#19101
DNA ladder	Thermo Scientific	#SM0311
DNA sample dye	Thermo Scientific	#R0611
Lipopolysaccharide (LPS) (TLR4 Agonist - Ultrapure LPS from E. coli 055:B5)	InvivoGen	#tlrl-pb5lps
JQ1	Sigma-Aldrich	#SML0974
Macrophage colony-stimulating factor (M-CSF)	R&D Systems	#416-ML-010/CF
Protease inhibitor cocktail	Sigma-Aldrich	#11697498001
Protein A agarose beads/ Salmon sperm DNA 2.5 mL	Sigma-Aldrich	# 16-157
Dream Taq PCR Master Mix	Thermo Scientific	#K1081
SYBR Green Master Mix	Roche	#04707516001
Critical commercial assays
PCR purification kit	Qiagen	#28104
cDNA kit	Thermo Scientific	#K1622
Bradford assay reagent	Bio-Rad	#500-0006
Experimental models: Cell lines
Bone-marrow-derived macrophages (BMDMs)	BALB/c mice (4–6 weeks, female)	N/A
Experimental models: Organisms/strains
L. donovani	ATCC	MHOM/IN/1983/AG83; ATCC repository number-PRAR-413
Oligonucleotides
Primers flanking the super enhancer element of miR146a-5p Forward: GTCTTGCTGAGGAGGTG Reverse: AGACGAGCTGCTTCAAGT	GCC Biotechnology	N/A
Software and algorithms
Image Lab software	Bio-Rad	N/A
LightCycler software	Roche	N/A
GraphPad software	GraphPad Software Inc	N/A
Other
Rocker	Eppendorf	N/A
DNA gel tank	Bio-Rad	N/A
Cooled tabletop centrifuge	Eppendorf	N/A
22°C incubator	Thermo Scientific	N/A
37°C CO2 incubator	Thermo Scientific	N/A
Fisherbrand™ Sound Enclosure for Model 50 and 120 Sonic Dismembrator	Fischer Scientific	N/A
Vertiti 96-well Thermal cycler	Applied Biosystems	N/A
Light Cycler 96	Roche	N/A
GelDoc-XR ®	Bio-Rad	N/A
Vortex	Tarson	N/A
Multiskan Ex	Thermo Scientific	N/A
Nanodrop™ 2000	Thermo Fisher	N/A

Materials and equipment

20 mM phosphate buffered saline (pH-7.5)- 500 mL

Components	Amount added (g)	Final concentration (mM)
NaCl	4	137
KCl	0.1	2.7
Na2HPO4	0.7	8.0
KH2PO4	0.1	1.5

Add all the above salts in 800 mL of MiliQ H₂O. After adjusting the pH of buffer up to 7.5 using 12 N HCl the solution volume is made up to 1 lit. After filter sterilization PBS can be stored at 4°C for 2–4 months.

Name of the buffer	Components	Amount added (g)	Final concentration (M)	pH – adjusted using
HEPES-KOH (100 mL)	HEPES free acid	23.8	1.0	7.5–1 N KOH solution
EDTA buffer (100 mL)	EDTA free acid	29.22	1.0	8.0–1 N NaOH
Tris-Cl (100 mL)	Tris base	12.114	1.0	8.0–12 N HCl

Note: Always make sure that ∼500 mL of 20 mM phosphate buffered saline (PBS, pH-7.5), ∼100 mL of 1 M HEPES-KOH (pH-7.5), 1 M EDTA buffer (pH-8.0) and 1 M Tris-Cl (pH- 8.0) are prepared beforehand as mentioned below. While preparing the buffers make sure the pH is adjusted before volume makeup with MilliQ as required.

Stock reagents for RIPA, FA lysis buffer and wash buffers

Components	Amount added	Volume of water (mL)	Stock concentration
Triton-X 100	1 g	10	10%
Na-deoxycholate	1 g	10	10%
Sodium Dodecyl Sulfate (SDS)	1 g	10	10%
Nonidet P-40 (NP-40)	1 mL	10	10%

Final buffer preparation

FA lysis buffer

Reagent	Final concentration	Stock concentration	Add to 500 mL
HEPES-KOH pH 7.5	50 mM	1 M	25 mL
NaCl	140 mM	1 M	70 mL
EDTA pH 8.0	1 mM	1 M	0.5 mL
Triton X-100	1% (v/v)	10% (v/v)	50 mL
Na-deoxycholate	0.1% (w/v)	10% (w/v)	5 mL
SDS	0.1% (w/v)	10% (w/v)	5 mL
MilliQ			344.5 mL

Filter sterilize FA Lysis Buffer and store at 4°C for 7–10 days. Just ∼20 min before use add 10 μL complete protease inhibitor cocktail (as purchased from supplier) per 10 mL of buffer.

RIPA Buffer

Reagent	Final concentration	Stock concentration	Add to 500 mL
Tris-Cl pH 8.0	50 mM	1 M	25 mL
NaCl	150 mM	1 M	75 mL
EDTA	2 mM	1 M	1 mL
Nonidet P - 40	1% (v/v)	10% (v/v)	50 mL
Na-deoxycholate	0.5% (w/v)	10% (w/v)	25 mL
SDS	0.1% (w/v)	10% (w/v)	5 mL
MilliQ			319 mL

Filter sterilize RIPA Buffer and stored at 4°C for 1–2 months. Just ∼20 min before use add 10 μL complete protease inhibitor cocktail (as purchased from supplier) per 10 mL of buffer.

Wash Buffer 1

Reagent	Final concentration	Stock concentration	Add to 500 mL
Tris-Cl pH 8.0	20 mM	1 M	10 mL
NaCl	150 mM	1 M	75 mL
EDTA	2 mM	1 M	1 mL
Triton X-100	1% (v/v)	10% (v/v)	50 mL
SDS	0.1% (w/v)	10% (w/v)	5 mL
MilliQ			359 mL

Store at 4°C for 7–10 days. Just before use add 10 μL complete protease inhibitor cocktail (as purchased from supplier) per 10 mL of buffer.

Wash Buffer 2

Reagent	Final concentration	Stock concentration	Add to 500 mL
Tris-Cl pH 8.0	20 mM	1 M	10 mL
NaCl	500 mM	1 M	250 mL
EDTA	2 mM	1 M	1 mL
Triton X-100	1% (v/v)	10% (v/v)	50 mL
SDS	0.1% (w/v)	10% (w/v)	5 mL
Milli Q			184 mL

Store at 4°C for 7–10 days. Just before use add 10 μL complete protease inhibitor cocktail (as purchased from supplier) per 10 mL of buffer.

Elution Buffer

Reagent	Final concentration	Stock concentration	Add to 500 mL
Sodium bicarbonate	100 mM	1 M	50 mL
SDS	1% (w/v)	10% (w/v)	50 mL
MilliQ			400 mL

Store at 37°C. Just before use add 10 μL complete protease inhibitor cocktail (as purchased from supplier) per 10 mL of buffer.

Other solutions

Name	Reagents
M199 media	100 mL media supplemented with 10% fetal bovine serum and 100 U/mL penicillin, 100 μg/mL streptomycin antibiotic solutions. It can be stored at 4°C for 4–6 months.
Dulbecco’s modified eagles’ media	100 mL media containing 4.5% D-glucose, 10% fetal bovine serum and 100 U/mL penicillin, 100 μg/mL streptomycin antibiotic solutions. It can be stored at 4°C for 4–6 months.
M-CSF	10 μg/mL stock as purchased from supplier. Store at −20°C.
LPS	1 mg/mL working stock in serum-free DMEM from 10 mg/mL stock as purchased from supplier. Store at −20°C.
JQ1	100 μM working stock of JQ1 prepared in 1 mL cell-culture grade DMSO. Store at 4°C.
Paraformaldehyde	4% (w/v) solution in PBS.
Glycine	1 M working solution prepared before use.
TAE	1 L of 50× stock contains 242 g Tris base, 57.1 glacial acetic acid, 100 mL 0.5 M EDTA (pH 8). It can be stored at 37°C for 8–12 months.
Salmon Sperm DNA	10 mg/mL as purchased from supplier. Store at −20°C.

Step-by-step method details

Macrophage infection and stimulation

Timing: 4–24 h

Stimulation or infection of mouse macrophages is done to induce super-enhancer (SE)- mediated miRNA transcription

• 1.Passage ∼2–3 × 10⁶ differentiated macrophages on 6-well culture dish (30 mm diameter) in M-CSF free 2 mL DMEM per well, after 6 days of stimulation with M-CSF.
• 2.Stimulation-24 h after plating, maintain macrophages either as untreated group (Figure 2A) or treat with stimulants, either 100 ng/mL LPS as positive control or 500 nM (final concentration) BET protein inhibitor JQ1 as negative control.
• 3.In case of positive control set, incubate macrophages in the presence of LPS containing media for 24 h (Figure 2B).
• 4.
  In case of negative control set, first incubate the macrophages in serum free media for 24 h.

  • a.
    Treat the macrophages with JQ1 for 2 h.
  • b.
    Remove the JQ1 containing media by washing with PBS once.
  • c.
    Incubate the cells for further 24 h in presence of serum supplemented DMEM (Figure 2C).

CRITICAL: It is critical to check that the stimulants are mixed well with media, as they are dissolved in DMSO.

• 5.Infection- Infect the macrophages with metacyclic stage of L. donovani promastigotes (Day 7 culture) for at least 4 h, followed by washing off of the extracellular promastigotes with sterile PBS for three times. 24 h post-infection, proceed with the infected cells for cross-linking step.

CRITICAL: Removal of extracellular parasites should be done thoroughly as non-phagocytosed parasites can affect the total cell and chromatin content of the sample.

Figure 2.

Differentiation and stimulation of BMDMs for X-ChIP

(A) Mouse bone marrow derived cells BMDMs, completely differentiated on Day 6 of mM-CSF stimulation.

(B and C) Images of BMDMs after stimulation with LPS and JQ1 for 24 h.

Cross-linking, quenching and lysis

Timing: 30 min

Cross-linking of cell chromatin is done to stabilize the miRNA SE complex followed by lysis of macrophages

• 6.Cross-linking- After completion of stimulation or infection, proceed to cross-linking of cell chromatin with sterile and freshly prepared paraformaldehyde solution.
• 7.Add 4% paraformaldehyde slowly and drop-wise in the media in order to reach the final concentration of paraformaldehyde to 0.75% in the media and incubate for 10 min at 37°C (troubleshooting 1).
• 8.While cross-linking always keep the 6-well plates under mild rotating condition (1,000–1,500 × g) for ensuring proper mixing of cross-linker with media.

CRITICAL: It is critical to gradually add the cross-linker, as sudden introduction of cross-linker may induce local and inadequate cross-linking which can affect the outcome/result of ChIP.

• 9.Quench the formaldehyde by adding glycine solution (final concentration of 125 mM in media) drop-wise under rotating condition and incubate the cells for 5 min at 37°C (troubleshooting 2).
• 10.As macrophages are adherent cells, remove the solution containing media directly from plate by pipetting and wash the cells twice with chilled PBS to remove any residual formaldehyde-glycine solution. Washing should be done by keeping the plate on rocking shaker with speed of 1,000–1,500 × g.
• 11.During second washing step, scrape the macrophages in presence of 1 mL 1× sterile PBS and pull together from three wells (2–3 × 10⁶/well) in 15 mL falcon tube. Centrifuge the cells at 5,000 × g for 3 min at 4°C under fixed angle rotor.
• 12.After removal of spent PBS, add around 750 μL of FA lysis buffer to ∼10 × 10⁶ macrophages per sample and incubate for 10 min on ice, followed by sonication.

Note: We have preferably used ready-made DMEM high glucose media with phenol red to track proper cross-linking. Presence of phenol-red helps in tracking cross-linking step by paraformaldehyde when it induces pH-dependent color change of media (from red to yellow). This color change takes place immediately after addition of paraformaldehyde. However, the protocol has been successfully tested upon cells cultured in the phenol red negative DMEM as well as RPMI media.

We have standardized and achieved controlled chromatin cross-linking for long SE element by modification of a previously published cross-linking kinetics (CLK) methodology to extract chromatin binding kinetics of protein in a time dependent manner.⁶ They reported that for 1% formaldehyde quenching 250 mM glycine was optimum.

Instead of time-kinetics, we performed paraformaldehyde-glycine concentration dependent kinetics. Here we used 0.5,0.75,1,1.5% of paraformaldehyde subsequently to cross-link the cell chromatin for 10 min at RT. Then quenched them with 125 mM glycine. It was found that at 1.5% paraformaldehyde concentration, macrophages got completely damaged and no pellet was rescued after PBS washing. Therefore, we used concentration below 1%, that is 0.75% paraformaldehyde-125 mM glycine for controlled cross-linking.

Sonication and protein estimation

Timing: 20–45 min

Sonication and protein estimation of lysate isolated from cross-linked macrophages

• 13.
  ∗Sonication.

  • a.
    Sonicate the cell lysates using 10 cycles of 50% amplitude 350 Hz) pulse for 15 s and 45 s intervals in ultrasound sonication instrument (troubleshooting 3).
  • b.
    During sonication keep the samples constantly on ice (troubleshooting 4).
• 14.Besides samples, sonicate around 1,500 μg/μL Salmon Sperm DNA (stock-10 mg/mL) in presence of 200 μL RIPA buffer which will be further used for coating beads. Sonicated salmon sperm DNA can be stored at −20°C for 2–4 months.
• 15.
  After sonication, centrifuge the lysate at 8,000 × g for 30 s at 4°C fixed angle rotor to remove residual cell debris. Transfer the clear supernatant in fresh sterile eppendorfs.

  • a.
    ∼100 μL input solution was kept separated as INPUT for DNA purification using Qiagen PCR purification kit. DNA purification should be carried out following the standard manufacturer’s protocol provided along with the kit.
  • b.
    Elute the purified DNA using 40 μL of nuclease free water. The isolated DNA can be kept at −20°C for nearly 2–4 weeks.
  • c.
    Mix 5 μL purified sample with1 μL of 6× DNA gel loading dye and resolve in 1% agarose gel using electrophoresis. Run the agarose gel 60–70 V speed in 1× TAE buffer. Subsequently stain the gel in Ethidium bromide and visualize using UV-transillumination of GelDoc-XR ® system (Biorad).
  • d.
    Run 1 kb DNA ladder alongside the samples in the same gel to understand the size of the fragmented chromatin DNA.

Note: The carcinogenic effect of Ethidium bromide can be avoided by alternative DNA staining chemicals such as SYBR™ Safe DNA Gel Stain (Invitrogen) or Gel Red™ solution (Biotium).

Due to large length of the SE elements compared to normal enhancers, standardization of the ultrasonic sound-based sonication step on cool platform is done as follows in order to achieve sheering of the chromatin into 500–1,000 kb size (Figure 3).

Figure 3.

Standardization of sonication cycle and cross-linker percentage for X-ChIP

(A) PCR purified DNA resolved in 1% agarose and visualized using EtBr staining.

(B) Cycles of ON/OFF pulses applied to standardize the optimum sheering of genomic DNA for X-ChIP.

After sheering the chromatin, isolate the DNA from supernatant by PCR purification kit as mentioned above and resolve 5 μL sample in 1% agarose gel for sizing of the DNA fragments (Figure 3).

As per sonication results, 15 s ON/ 45 s OFF pulse with 50% amplitude produced best result with clear 500–1,000 bp DNA fragmentation and therefore we recommend using this optimized sonication cycle for SE element X-ChIP assay (Figure 3B).

Bead preparation and antibody-chromatin incubation

Timing: 45 min–24 h

Preparation of beads for affinity purification is done followed by generation of antibody-chromatin complex

• 16.Measure protein content of the samples by Bradford Assay reagent.
• 17.During Bradford assay, dilute the 5× Bradford reagent in sterile water to obtain 1× solution and incubate 98 μL reagent with 2 μL of blank (Water) or standards (BSA solutions 0.00, 0.25,0.5 and 1 mg/mL) or samples (untreated, LPS treated, JQ treated and infected). Record the colorimetric reading at 595 nm using the Multiskan-Ex reader. Therefore, prepare a standard curve in order to obtain the final concentration of all the samples (Figure 4).
• 18.After separating INPUT, dilute the sample (from step 22) 100 times with around 500 μL RIPA buffer in order to obtain around 25 μg total protein before incubating with pre-coated beads and antibodies.
• 19.Dilute each sample to obtain 25 μg total protein for incubating with pre-coated beads and antibodies.
• 20.
  Bead pre-coating.

  • a.
    For each sample, take at least 20 μL Protein A agarose bead and wash three times with RIPA buffer by centrifugation at 4,000 × g for 10 min at 4°C temperature (troubleshooting 5).
  • b.
    After removal of supernatant, resuspend the beads in 330 μL of RIPA.
  • c.
    Mix ∼75 ng/μL bead volume of sonicated salmon sperm DNA with 20 μL of beads along with 0.1 μg/mL BSA (prepared in PBS). So, from 1,500 μg/μL sonicated salmon sperm DNA stock (from step 15c), add 20 μL bead along with 660 μL RIPA buffer (3× of total volume) and incubate for 30 min in orbital rotor (100–150 × g speed) at 4°C.
  • d.
    After coating, remove the supernatant by centrifugation at 4,000 × g for 10 min followed by two times washing with 500 μL RIPA buffer at 4°C.
  • e.
    Keep the ratio of bead: antibody: protein (lysate) as 20:10:25 i.e., with 20 μL pre-coated beads, mix 10 μg of antibody solution (1 mg/mL stock) and 25 μg protein lysate. Incubate the suspension for 24 h in orbital rotor (250–300 × g speed) at 4°C (troubleshooting 6).

Note: Protein concentrations obtained are provided in the expected outcomes section. Estimation of protein concentration is crucial as lower amount of protein can give negative result and over concentrated antigen in the binding reaction might end up giving false positive outcome of ChIP.

Figure 4.

BSA standard curve obtained by Bradford assay

(A) Optical Density values for BSA solutions (mentioned concentrations in mg/ml) obtained at 595 nm(OD₅₉₅) were plotted as standard curve.

(B) OD₅₉₅ of Table represnting the concnetrations of all the samples that were calculated from the BSA standard curve.

Bead coating can be performed 1–2 days prior starting ChIP and kept at 4°C in RIPA buffer.

Standardization of antigen-antibody interaction

We have standardized the antibody: antigen ratio in order to obtain the optimized condition to pull down the specific SE element (Table 2). The effect of different antigen and antibody ratio upon the quality of the X-ChIP can be found in Figure 5A.

Table 2.

Antigen and antibody ratio optimization for SE region pull down

Ratio (Ab: Ag)	Antibody (μg)	Antigen/lysate (μg)
1:5	1	5
5:10	5	10
10:25	10	25
25:80	25	80

Figure 5.

Analysis of BRD4 accumulation in SE element of miRNA encoding gene

(A and B) The alteration of BRD4 accumulation according to the different antibody and antigen ratio and (B) different stimulation conditions- US-unstimulated, INF- infected, LPS- Lipopolysaccharide and JQ1 treated macrophages.

(C) Semi-quantitative PCR of BRD4-pulled miR146a-5p SE DNA. Percent accumulation of BRD4 protein upon miR146a-5p SE elements on- US-unstimulated, INF- infected, LPS- Lipopolysaccharide and JQ1 treated macrophages. Each experiment has been performed in triplicates. Statistical significance (∗∗P<0.01, ∗∗∗P<0.001) was calculated using one-way ANOVA and data are represented as mean± SD (Graph pad prism 5.0).

Washing and elution

Timing: 1–2 h

Washing of bead-antibody-chromatin complex and elution of bound SE DNA from complex

• 21.Next day, centrifuge the bead and antigen-antibody containing suspensions at 2,000 g speed for 1 min at 4°C and remove the supernatant.
• 22.Wash the beads three times with wash buffer 1.
• 23.Then wash the beads with wash buffer 2 once before proceeding to the elution step.
• 24.For each wash, keep the suspension on shaker for 3 min at 4°C and centrifuge at 4,000 × g speed for 1 min to remove the wash solution.

Note: Wash by gentle vortex (for around 5–10 s). Strictly do avoid pipetting to prevent rupture and damage of the agarose beads.

• 25.Elute the DNA from the bound/pulled down chromatin from beads by adding 120 μL of elution buffer to the protein A beads and rotate the mixture for 15 min at 37°C.
• 26.Then centrifuge the solution at 2,000 g speed for 1 min and collect the supernatant in fresh tube for DNA purification.

Note: DNA is purified from both INPUT (mentioned in step-20) and pulled down solutions. Before DNA purification, 0.5 mg/mL RNase A is added in each suspension.

• 27.Using Qiagen PCR purification kit, purify DNA from each set and measure the concentration of DNA by Nanodrop (Table 3).

Table 3.

Concentration of DNA measured by Nanodrop

Sample no.	DNA (ng/μL)	X for 10 ng = DNA (μL)
US	35	0.28
INF	40	0.25
LPS	50	0.20
JQ1	25	0.40
1:5	50	0.20
5:10	45	0.23
10:25	40	0.25
25:80	55	0.18

PCR

Timing: 3 h

Polymerase chain reaction-based amplification of miR146a-5p SE region

• 28.Perform semi-quantitative PCR and quantitative RT-PCR using ∼10 ng INPUT DNA as well as pulled down DNA as templates.
• 29.Resolve semi-quantitative PCR products in 1.5% agarose gel supplemented with 10 μg/mL ethidium bromide in presence of 1× TAE buffer at 70 V. Document the gels using UV-transilluminator system of GelDoc-XR.
• 30.Calculate the accumulation kinetics of target protein (in our model, BRD4) upon SE element from the Ct values obtained from RT-PCR and represent it as percent accumulation. Calculation has been explained in the expected outcomes section (Figure 5B and Table 4).

PCR reaction master mix for semi-quantitative PCR reaction

Reagent	Volume
DNA template (10 ng)	X (μL as per Table 3)
Dream Taq master mix (2×)	5.0 μL
Primer Forward (25 μM)	1.0 μL (25 nM)
Primer Reverse (25 μM)	1.0 μL (25 nM)
Total	Adjust volume upto 20 μL

Details of semi-quantitative PCR reaction cycle performed in Thermal cycler (Thermo Scientific)

Steps	Temperature	Time	Cycles
Initial Denaturation	95°C	60 s	1
Denaturation	95°C	10 s	25 cycles
Annealing	48°C	30 s
Extension	72°C	30 s
Final extension	72°C	7 min	1
Hold	4°C	Forever

RT-PCR reaction master mix for quantitative PCR reaction

Reagent	Amount
DNA template (10 ng)	Previously mentioned
SYBR Green master mix (2×)	5.0 μL
Primer Forward (25 μM)	1.0 μL (Final 25 nM)
Primer Reverse (25 μM)	1.0 μL (Final 25 nM)
ddH2O	Accordingly, to make final volume
Total	10 μL

Details of RT- PCR reaction cycle performed in Light Cycler 96

Steps	Temperature	Time	Cycles
Preincubation	95°C	600 s	1
Denaturation	95°C	30 s	40 cycles
Annealing	48°C	30 s
Extension	72°C	60 s
Melting	95°C	10 s	1
Melting	65°C	60 s	1
Melting	97°C	1 s	1
Cooling	37°C	30 s	1

Table 4.

Calculation of percent input (% BRD4 accumulation in SE of miR146a-5p) and possible outcomes

Samples	CHIP DNA Ct1	CHIP DNA Ct2	Input Ct1	Input Ct2	Adj input Ct1	Adj input Ct2	Adj input 1 - CHIP DNA 1	Adj input 2 - CHIP DNA 2	% Input 1	% Input 2
Uninfected	39.24	38.95	31.36	32.3	24.716	25.656	-14.524	-13.294	0.004245	0.009957
Infected	30.24	31.27	29.1	29.88	22.456	23.236	-7.784	-8.034	0.453714	0.381527
LPS	29.6	29.2	30.86	31.2	24.216	24.556	-5.384	-4.644	2.394719	3.999601
JQ1	37.38	38.45	31.55	31.1	24.906	24.456	-12.474	-13.994	0.017577	0.006129
Ab:Ag (1:5)	30.74	30.35	30.54	30.33	23.896	23.686	-6.844	-6.664	0.87046	0.986134
Ab:Ag (5:10)	30.42	30.03	30	30.11	23.356	23.466	-7.064	-6.564	0.74735	1.056913
Ab:Ab (10:25)	29.99	29.5	31.3	31.27	24.656	24.626	-5.334	-4.844	2.479168	3.4102
Ab:Ab (15:50)	29.82	29.8	30.7	31.6	24.056	24.956	-5.764	-4.844	1.840192	3.481855

Expected outcomes

The present protocol is a modified version of chemical cross-linked ChIP, that will be specifically used for pulling down long length SE element of miRNA gene. The protocol can pull down BRD4 occupied SE element that controls transcription of miR146a-5p encoding exon. Pull-down of SE element has been performed using macrophages stimulated LPS (positive control), JQ1 (BRD4 inhibitor and negative control) and L. donovani infection (Figure 2). Our protocol mainly could retrieve long and complex super-enhancer (SE) domains with controlled cross-linking and sonication (Figure 3). The percent input of BRD4 indicated that during infection or LPS stimulation BRD4 occupied the SE element of miR146a-5p and data. A clear BRD4 occupancy profile at miRNA SE element has been obtained using this current protocol (Figure 5C). This protocol can be applied for other longer enhancer elements as well with similar binding proteins and kinetics.

Quantification and statistical analysis

Protein concentration of lysate measured by Bradford

i. BSA standard curve

BSA standard concentration (mg/mL)	OD595 nm
0.00	0
0.125	0.016
0.25	0.036
0.5	0.089
1.0	0.1255

ii. Protein concentration

Sample No	Concentration (mg/mL)
US	2.26
INF	2.19
LPS	2.39
JQ1	2.75
1:5	2.24
5:10	2.16
10:25	2.15
15:50	2.27

Calculation of percent accumulation of protein upon SE element of miRNA

The percent input method is followed to calculate the kinetics of target protein accumulation upon super enhancer DNA. The method has following steps of calculations (Figure 6).

• 1.Use 1% of the initial input for performing antibody based pull down experiment so the dilution factor (DF) is log₂ 100 = 6.64.
• 2.Subtract DF from the Ct value of the input to obtain the Adjusted input (Adj input Ct).

Adj input Ct= (Raw input Ct- 6.644)

• 3.Calculate the final percent accumulation as followed.

Percent input = 100∗2ˆ (Adj input Ct- ChIP DNA Ct)

• 4.Finally plot the percent input values and represent them as bar diagrams using graph pad prism software (statistical analysis details are given below).

Figure 6.

Flow chart explaining calculation of percent protein accumulation upon SE element

Limitations

The protocol has not been yet applied for any blood or tissue sample. Using paraformaldehyde and glycine quencher solution in whole blood may involve change in pH and chemical hurdles for obtaining controlled cross-linking. Due to limitation of using the current sonication protocol in tissue samples the protocol may not be successful in isolating SE region related to tissue samples. Our current protocol is capable of rescuing chromatin fragments of length till 1 kb. Obtaining larger chromatin fragment may not be possible using this current version of protocol and may need more improvisation in the sonication step.

Troubleshooting

Problem 1

Less or no PCR product after antibody pull-down which may be due to inadequate cross-linking of chromatin with large SE complex components (related to step 7).

Potential solution

User has to strictly maintain the percentage and time of cross-linking within 0.75%- and 15 min window respectively.

Problem 2

False positive results showing unexpected occupancy of SE protein on chromatin element is indicative of over-cross-linking (related to step 9).

Potential solution

Besides this the user has to be careful about concentration of the cross-linking agent (here 125 mM Glycine) and time of cross-linking which is 5 min in this protocol.

Problem 3

The sonication step has been modified to obtain a controlled shearing of chromatin DNA. Users may get smeared DNA which indicates over-fragmentation of DNA, even after using the recommended cycle (related to step 13a).

Potential solution

The instrument output (as amplitude) has to be checked and has to be within the range (∼350 Hz). High output may result in high current infusion even using same ON/OFF cycle.

Problem 4

Even after proper fragmentation, user may not get PCR product after pull down with antibody against target protein. This may occur due to disruption of transcription factor complex that occupy the SE region (related to step 13b).

Potential solution

The probe and DNA suspension should be constantly placed inside ice else the heat generated during sonication may damage the DNA fragments and also disturb the protein complex occupying the chromatin section.

Problem 5

Sometimes the type of bead used affects the affinity and efficiency of SE pull down and results in non-specific pull down of chromatin sections. This mostly arises due to differential affinity of beads for different isotypes of antibodies (related to step 20a).

Potential solution

Here we have used protein A beads as it reportedly has high affinity for rabbit monoclonal antibodies. Protein G beads are highly recommended for efficient pull-down using mouse monoclonal antibodies. Users should use protein A/G beads which provide efficiency during pull down using polyclonal antibodies.

Problem 6

Unsuccessful or negative pull-down results in PCR after this step can be due to the fact that the epitopes of antigen with which the antibody binds are accidentally shared by the DNA binding domain of the antigen (in our case BRD4) and therefore get masked (related to step 20e).

Potential solution

Authors are suggested to cautiously choose the antibody for pull down step and if necessary, change the clone of the antibody for pull down which can bind a different epitope of antigen.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by Prof. Nahid Ali, email- nali@iicb.res.in.

Materials availability

This study didn’t generate any new unique reagents.

Data and code availability

This study did not generate/analyze [datasets/code].