Native Chromatin Preparation and Illumina/Solexa Library Construction

INTRODUCTION

High-throughput whole-genome analysis has become a practical and important technique to understand nuclear processes such as transcription, replication and genome structure. Though microarrays have been the preferred genome-scale analysis method for over a decade, new technologies, referred to as next-generation sequencing offer distinct advantage over microarrays in sensitivity and scale (Schones and Zhao, 2008). Currently, several next-generation sequencing platforms are available, including the Genome Analyzer (Solexa/Illumina), 454 (Roche) and ABI-SOLiD (Applied Biosystems). These platforms can be used for a variety of studies such as sequencing of chromatin immunoprecipitated DNA (ChIP-Seq) (Barski et al., 2007; Johnson et al., 2007; Mikkelsen et al., 2007; Robertson et al., 2007), nucleosome position profiling (Schones et al., 2008), DNA methylation studies (Cokus et al., 2008), transcriptome profiling (RNASeq) (Mortazavi et al., 2008; Nagalakshmi et al., 2008) and SNP genotyping. The following protocol describes sample preparation for ChIP-Seq to analyze histone modification patterns using native chromatin and the Genome Analyzer (Solexa/Illumina) (Barski et al., 2007; Wang et al., 2008). One advantage of using native chromatin as compared to cross-linked chromatin is that it provides single nucleosome level resolution and avoids non-specific modification signals from different nucleosomes carried over through protein-protein interactions. The protocol includes purification of human CD4⁺ T cells from lymphocytes and chromatin fragmentation using microccocal nuclease (MNase) digestion, followed by chromatin immunoprecipitation (ChIP) and construction of a library for sequencing.

MATERIALS

Reagents

• CD4⁺ T cell Isolation kit II (human) (Miltenyi Biotec)

• DNA END-Repair kit (Epicentre Biotechnologies)

• Dynabeads Protein A (Invitrogen)

• Ethanol (70% and 100%)

• Gel extraction kit (QIAGEN)

• Glycerol

• Glycogen, 20 mg/ml

• LiCl buffer (0.25 M LiCl, 0.5% NP-40, 0.5% Na deoxycholate)

• Lymphocyte separation medium (LSM, Cambrex Bio Science)

• Lymphocytes, human leukapheresis pack from a blood bank

• Micrococcal nuclease (MNase) from Staphylococcus aureus (Sigma)

• MNase digestion buffer (50 mM Tris-HCl, pH 7.6, 1 mM CaCl₂, 0.2% Triton X-100 or NP-40, 5 mM butyrate, 1x proteinase inhibitor cocktail, and 0.5 mM fresh PMSF)

• MNase stop buffer (10 mM Tris, pH 7.6, 10 mM EDTA-final concentration)

• NaCl

• NaOAc (3 M, pH 5.3)

• PBS (1X)

• Phenol-CHCl₃ (1:1)

• Proteinase K (Roche Diagnostics).

• QIAquick PCR purification kit (QIAGEN)

• RIPA buffer (10 mM Tris, pH 7.6, 1 mM EDTA, 0.1% SDS, 0.1% Na-deoxycholate, 1% Triton X-100)

• SDS (10%)

• Solexa PCR primers

• T cell isolation buffer (1X PBS containing 0.5 % BSA, filtered)

• T4 DNA ligase (400 units/μl) and 10X buffer

• Taq polymerase (New England Biolabs)

• TE (1X), pH 7.4

• Triton-X100

Equipment

• Agarose gels (2%)

• E-Gels (2%)-Invitrogen

• LS column (Miltenyi Biotec)

• Magnetic stand for isolation of beads

• Microcentrifuge tubes

• Spectrometer, Qubit

• Test tubes, 50-ml conical and 250-ml conical

METHOD

Isolation of human CD4⁺ T Cells using CD4⁺ T Cell Isolation Kit II (Human) (Miltenyi Biotec)

Note: To obtain best results it is important to carry out steps 1 to 27 on the same day.

1. On the day of the experiment obtain leukapheresis pack, which contains leukocytes enriched by apheresis from human peripheral blood, from a blood bank. Dilute 10⁹ leukocytes with 1X PBS (1:1). Pipette 15 ml lymphocyte separation medium (LSM) in a 50-ml conical tube. Carefully layer lymphocyte suspension over the LSM. A clear boundary between the two phases is critical for the removal of erythrocyte contamination.

2. Centrifuge at 2000 rpm for 25 min at 20°C. After centrifugation, the order of layers from top to bottom is plasma/platelets, lymphocytes, Ficoll-Hypaque, and granulocytes/erythrocytes.

3. Aspirate off the top layer and leave the lymphocyte interphase intact. Transfer the cells in the interphase to a 250-ml conical tube. Wash with 1X PBS and centrifuge at 800 rpm for 12 min at 20-23°C to remove the residual platelets. Remove supernatant, resuspend cells in 1X PBS and repeat the wash once to remove remaining platelets.

4. Resuspend in 20-30 ml 1X PBS and then count the cells. Aliquot 0.5 × 10⁶ cells for FACS analysis.

Magnetic labeling of non-CD4⁺ T Cells

• Keep the cells cold

• 5Take 10⁸ cells from Step 4

Note: The final yield of purified CD4⁺ T Cells is approximately 20%.

• 6Centrifuge the cell suspension at 300×g for 10 min.
• 7Resuspend the cell pellets in 40 μl of T cell isolation buffer per 10⁷ cells.
• 8Add 10 μl of biotin-antibody cocktail (from T cell isolation kit) per 10⁷ cells.

Note: The biotin-antibody cocktail contains monoclonal antibodies against CD14, CD16, CD19, CD36, CD56, CD123 and Glycophorin A.

• 9Mix well and incubate at 4-8°C or on ice for 10 min.
• 10Add 30 μl of T cell isolation buffer per 10⁷ cells.
• 11Add 20 μl anti-biotin microbeads (from T cell isolation kit) per 10⁷ cells.
• 12Mix well and incubate at 4-8 °C for 15 min.
• 13Wash cells with T cell isolation buffer by adding 10-20× the final labeling volume. Centrifuge at 300×g for 10 min. Pipette off supernatant completely.
• 14Resuspend the cells completely into appropriate volume of T cell isolation buffer. Use 3 ml for 250-300 million cells.
• 15Proceed to magnetic separation using an LS column (Step 16).

LS Column Separation – Depletion of non-CD4⁺ T Cells

• 16Place column in the magnetic field of a suitable MACS separator.
• 17Prepare the column by rinsing with 3 ml T cell isolation buffer. The buffer will run through the column quickly.

Note: This step can be performed during antibody incubation (Step 12).

• 18Apply cell suspension onto the column: load ~250 to 300 million cells per column. Allow the cells to pass through and collect effluent. This fraction contains unlabeled cells and represents the enriched CD4⁺ T cell fraction.
• 19Wash the column 3X with 3 ml of T cell isolation buffer. For each wash, add buffer once the column reservoir is empty. Collect the entire effluent in the same tube (Step 18)
• 20Count cell number and check CD4⁺ T cell purity. Recovery is typically about 20%.

Note: Purified CD4⁺ T cells with about 95%-98% purity are used for native chromatin preparation.

• 21Centrifuge at 300×g for 10 min.

Note: Alternatively, Dynabeads Untouched human CD4 T cells kit (Invitrogen) can be used.

Native Chromatin Preparation by MNase Digestion

Note: This step is only for analyzing histone modifications. For the analysis of the target sites of protein that may not be as stably or directly bound to the chromatin as the histones, the chromatin should be cross-linked using formaldehyde and sonicated to 200-to 300-bp length.

• 22Resuspend 20-22×10⁶ purified human CD4⁺ T cells from Step 21 in 1 ml MNase digestion buffer kept at room temperature and add protease inhibitors.
• 23Add 0.3U of MNase and incubate for 5 min at 37°C

Note: the amount of MNase used for each cell-type needs to be tested.

• 24Stop the reaction by adding Stop Buffer with the final concentration: 10 mM Tris, pH 7.6, 10 mM EDTA.
• 25Sonicate for 3 × 20 seconds in ice water.
• 26Dialyze against 400 ml of RIPA buffer for 2 hrs at 4°C.
• 27Centrifuge for 10 min at 4°C. Transfer supernatant to a new tube, add glycerol to a final concentration of 5%, and store at -80°C.
• 28To check the size of the isolated chromatin, transfer 20 μl of supernatant to a new tube. Add 4 ug of RNAse A and incubate for 5 min at room temperature. Add 3 μl of 10% SDS and 5 μl of 20 mg/ml proteinase K. Incubate at 65°C for 2 h and purify the DNA by phenol-CHCl₃ extraction and ethanol precipitation. Analyze on a 2% agarose gel.

Note: The above digestion procedure would primarily yield mononucleosomes. To obtain other fragment lengths the MNase amounts and digestion time should be varied.

• 29Continue to the ChIP experiment (Step 30).

Chromatin Immunoprecipitation (ChIP)

Note: Do NOT block beads with any non-specific DNA or RNA.

• 30Take 40 μl of Dynabeads Protein A in two microcentrifuge tubes. Wash with 600 μl 1X PBS. Place the tubes in a magnetic stand and aspirate off the PBS. Add 100 μl 1X PBS and 4 μg antibody or preimmune serum to the beads. Rotate for 4-6 h at 4°C.
• 31Using the magnetic stand, aspirate the supernatant and wash the beads twice with 0.2 ml 1X PBS to remove free IgGs (5 min each wash).
• 32Remove the supernatant from the beads. Add 500 μl of the chromatin extracts (from 10⁷ cells) to the beads. Rotate at 4°C overnight.
• 33
  Wash the beads (10 min each wash):

  • 2× with 1 ml of RIPA buffer
  • 2× with 1 ml of RIPA buffer + 0.3 M NaCl
  • 2× with 1 ml of LiCl buffer
  • 1× with 1 ml of 1X TE + 0.2% Triton X-100
  • 1× with 1 ml of 1X TE.

Note: To obtain less stringent washing, reduce the concentration of NaCl in RIPA buffer and/or reduce the washing with all the buffers to 1×.

• 34Resuspend the beads in 100 μl 1X TE. Add 3 μl of 10% SDS and 5 μl of 20 mg/ml proteinase K. Incubate overnight at 65°C.
• 35Vortex briefly and transfer the supernatant to a new tube using a magnetic stand. Wash the beads with 100 μl 1X TE and 0.5 M NaCl. Combine the supernatant. Extract once with 200 μl of phenol/chloroform. Add 2 μl of 20 mg/ml glycogen, 20 μl of 3 M NaOAc (pH 5.3), and 500 μl of ethanol to the supernatant to precipitate the DNA.
• 36Wash the pellet once with 70% ethanol.
• 37Resuspend the pellet in 50 μl of 1X TE. Use 2 μl of diluted (1:5) DNA for real-time PCR confirmation.

TaqMan Real-time PCR Analysis

• 38
  Before moving to the next step, perform real-time PCR to ensure that ChIP has worked well.

  	For 25 μl final volume	For 20 μl final volume
  2× Master mix	12.5 μl	10 μl
  Primer mix (5 μM each)	2.5 μl	2 μl
  Probe (2 μM)	2.5 μl	2 μl
  dd H2O	5.5 μl	4 μl
  DNA template:	2 μl	2 μl

DNA Repair and Solexa Library Construction

• 39
  Repair DNA ends to generate blunt-ended DNA using the Epicentre DNA END-Repair kit.

  • 1-34 μl DNA (0.3 mg)
  • 5 μl 10X End repair buffer
  • 5 μl 2.5 mM each dNTP
  • 5 μl 10 mM ATP
  • x μl H₂O to adjust the reaction volume to 49 μl
  • 1 μl End-Repair enzyme mix (T4 DNA Pol + T4 PNK)

• Incubate at room temperature for 45 min.

• 40Use MinElute PCR purification kit or phenol/chloroform extraction to purify DNA. Elute or resuspend DNA on 30 μl 1X TE, pH 7.4
• 41
  Add “A” to 3' ends.

  • 30 μl DNA from Step 40
  • 2 μl H₂O
  • 5 μl 10× Taq buffer
  • 10 μl 1 mM dATP
  • 3 μl 5 U/μl Taq DNA polymerase

• Incubate at 70°C for 30 min. Purify using QIAGEN MinElute PCR purification kit or phenol/chloroform extraction. Elute with or resuspend in 10 μl 1X TE, pH 7.4.

Note: Alternatively, Klenow fragment exo^- can be used for adding ‘A’ bases.

• 42
  Perform linker ligation.

  • 10 μl DNA (300 ng)
  • 9.9 μl H₂O
  • 2.5 μl 10X T4 DNA ligase buffer
  • 0.1 μl Adaptor oligo mix
  • 2.5 μl T4 DNA ligase (400 units/μl)

• Incubate at 20-23°C for 30 min and then at16°C overnight.

• 43Purify DNA using MinElute PCR purification kit. Elute with 20-25 μl buffer EB
• 44
  Size selection-2% E-Gel

  • Load 20 μl linker ligated DNA onto a 2% E-Gel (Invitrogen).
  • Cut the gel around the 200-400 bp region
  • Note: DNA would not be visible on the gel at this step. Use only one sample per gel to avoid contamination.
  • Extract the DNA using QIAGEN MinElute gel extraction kit. Elute with ~11.5 μl buffer EB.
• 45
  Amplify the DNA using Solexa primers and enzyme mix.

  • 10.5 μl of DNA
  • 12.5 μl of master mix
  • 1 μl of PCR primer 1 (2× diluted with 1X TE, pH 7.4)
  • 1 μl of PCR primer 2 (2× diluted with 1X TE, pH 7.4)
  • Total volume: 25 μl

• Denature at 98°C for 30 sec, followed by 18 amplification cycles (98°C, 10 sec; 65°C, 30 sec; 72°C, 30 sec.)

Note: Amplify for 18 cycles and then check 2.5 μl of product on a 2% agarose gel. If the band is not clearly visible, perform 3 more cycles and check again.

• 46
  Purify the amplified products

  • Load the amplified DNA on 2.5% agarose gel.
  • Excise the band at the 200-400 bp region.
  • Purify the DNA using Qiagen gel extraction kit.
  • Measure the DNA concentration using Qubit spectrometer.
  • The DNA can be used for cluster generation using the Illumina flow cells and sequencing-by-synthesis on Illumina Genome Analyzer following manufacturer's instructions.