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 advantages over microarrays in both sensitivity and scale. Several next-generation sequencing platforms are currently available, including the Genome Analyzer (Solexa/Illumina), 454 (Roche), and ABI-SOLiD (Applied Biosystems). This protocol describes sample preparation for sequencing of chromatin-immunoprecipitated DNA (ChIP-Seq) to analyze histone modification patterns using native chromatin and the Genome Analyzer. One advantage of using native chromatin as compared to cross-linked chromatin is that it provides single-nucleosome-level resolution and avoids nonspecific 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 micrococcal nuclease (MNase) digestion, followed by chromatin immunoprecipitation (ChIP) and construction of a library for sequencing.
RELATED INFORMATION
The advantages of next-generation sequencing have been described by Schones and Zhao (2008). These platforms can be used for a variety of studies, including 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 (RNA-Seq) (Mortazavi et al. 2008; Nagalakshmi et al. 2008), and SNP genotyping. Use of this protocol to analyze histone modification patterns is described in Barski et al. (2007) and Wang et al. (2008).
MATERIALS
Reagents
Adapter oligo mix (Illumina) (see Step 42)
Antibody and preimmune serum for ChIP
CD4+ T cell Isolation Kit II (human) (Miltenyi Biotec)
Alternatively, Dynabeads Untouched Human CD4 T Cells Kit (Invitrogen) can be used (see Steps 5-21).
DNA End-Repair Kit (Epicentre Biotechnologies) (see Step 39)
The kit includes end-repair enzyme mix, end-repair 10X buffer, dNTP solution, and ATP.
Dynabeads Protein A (Invitrogen)
Ethanol (70% and 100%)
Glycerol
Glycogen, 20 mg/mL
Klenow fragment exo- (optional; see Step 41.iii)
![\"recipe\"](\"dxy_expriment/201812/recipe.gif\")
LiCl buffer for ChIP
Lymphocyte separation medium (LSM)
Lymphocytes, human leukapheresis pack from a blood bank
The pack contains leukocytes enriched by apheresis from human peripheral blood.
Micrococcal nuclease (MNase) from Staphylococcus aureus (Sigma)
MinElute Gel Extraction Kit (QIAGEN)
The kit provides spin columns, buffers (including buffer EB), and collection tubes.
MinElute PCR Purification Kit (QIAGEN)
The kit provides spin columns, buffers (including buffer EB), and collection tubes.
MNase digestion buffer for ChIP
MNase stop buffer for ChIP
NaCl
NaOAc (sodium acetate) (3 M, pH 5.3)
![\"caution\"](\"dxy_expriment/201812/caution.gif\")
Phenol:chloroform (1:1)
Phosphate-buffered saline (PBS) (1X)
Phusion HF Master Mix (2X, Illumina) (see Step 45)
Polymerase chain reaction (PCR) primers PE 1.0 and PE 2.0 (Illumina) (see Step 45)
Primer mix for real-time PCR (see Step 38)
Several companies offer primer synthesis service.
Protease inhibitor cocktail for ChIP (1000X)
Proteinase K (20 mg/mL) (Roche Diagnostics)
Reagents for agarose gel electrophoresis
RIPA buffer for ChIP
RNase A
SDS (sodium dodecyl sulfate) (10%)
T cell isolation buffer (1X PBS containing 0.5 % bovine serum albumin, filtered)
T4 DNA ligase (400 U/µL) and 10X buffer
Taq polymerase (New England Biolabs)
TaqMan PCR master mix (Applied Biosystems) (see Step 38)
TE (1X, pH 7.4)
Triton X-100
Equipment
Agarose gels (2%) and electrophoresis equipment
Centrifuge
E-Gels (2%) (Invitrogen)
Ice water
LS column (Miltenyi Biotec)
MACS (magnetic-activated cell sorting) separator
Magnetic stand for bead isolation
Microcentrifuge tubes
Micropipettor and tips
Rotator
Sonicator
Spectrometer (Qubit Systems)
Test tubes, conical, 50- and 250-mL
Thermal cycler
Tubes (microcentrifuge)
Vortex mixer
Water baths or heating blocks preset to 16°C, 20°C-23°C, 37°C, 65°C, 70°C
METHOD
Isolation of Human CD4+ T Cells
To obtain best results, it is important to carry out Steps 1-27 on the same day.
Isolation of Lymphocytes 1. On the day of the experiment, obtain a leukapheresis pack from a blood bank. Dilute 109 leukocytes with 1X PBS (1:1). Pipette 15 mL of LSM into a 50-mL conical tube. Carefully layer the 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°C-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 of 1X PBS and then count the cells. Aliquot 0.5 x 106 cells for fluorescence-activated cell sorting (FACS) analysis. Magnetic Labeling of Non-CD4+ T Cells Use the CD4+ T Cell Isolation Kit II (human) (Miltenyi Biotec). Alternatively, Dynabeads Untouched Human CD4 T Cells Kit can be used for Steps 5-21. Keep the cells cold. 5. Aliquot 108 cells from Step 4. The final yield of purified CD4+ T cells is ~20%. 6. Centrifuge the cell suspension at 300g for 10 min. 7. Resuspend the cell pellets in 40 µL of T cell isolation buffer per 107 cells. 8. Add 10 µL of biotin-antibody cocktail (from T cell isolation kit) per 107 cells. The biotin-antibody cocktail contains monoclonal antibodies against CD14, CD16, CD19, CD36, CD56, CD123, and glycophorin A. 9. Mix well and incubate for 10 min at 4°C-8°C or on ice. 10. Add 30 µL of T cell isolation buffer per 107 cells. 11. Add 20 µL of anti-biotin microbeads (from T cell isolation kit) per 107 cells. 12. Mix well and incubate for 15 min at 4°C-8°C. 13. Wash cells with T cell isolation buffer by adding 10X-20X the final labeling volume. Centrifuge at 300g for 10 min. Pipette off supernatant completely. 14. Resuspend the cells completely into appropriate volume of T cell isolation buffer. Use 3 mL for 250-300 million cells. 15. Proceed to magnetic separation using an LS column (Step 16). LS Column Separation: Depletion of Non-CD4+ T Cells 16. Place LS column in the magnetic field of a suitable MACS separator. 17. Prepare the column by rinsing with 3 mL of T cell isolation buffer. The buffer will run through the column quickly. This step can be performed during antibody incubation (Step 12). 18. Apply cell suspension onto the column: load ~250-300 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. 19. Wash the column three times 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 (from Step 18). 20. Count cells and check CD4+ T cell purity. Recovery is typically ~20%. Purified CD4+ T cells with ~ 95%-98% purity are used for native chromatin preparation. 21. Centrifuge at 300g for 10 min. Native Chromatin Preparation by MNase Digestion
This step is only for analyzing histone modifications. To analyze target sites of proteins that may not be as stably or directly bound to chromatin as histones, the chromatin should be cross-linked using formaldehyde and sonicated to 200- to 300-bp length.
22. Resuspend 20-22 x 106 purified human CD4+ T cells from Step 21 in 1 mL MNase digestion buffer kept at room temperature and add protease inhibitor cocktail to 1X final concentration. 23. Add 0.3 U of MNase and incubate for 5 min at 37°C. Determine the amount of MNase to use for each cell type experimentally. 24. Stop the reaction by adding MNase stop buffer to a final concentration of 10 mM Tris (pH 7.6), 10 mM EDTA. 25. Sonicate three times for 20 sec each in ice water. 26. Dialyze against 400 mL of RIPA buffer for 2 h at 4°C. 27. Centrifuge 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. 28. To check the size of the isolated chromatin, transfer 20 µL of supernatant to a new tube. Add 4 µg 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:chloroform extraction and ethanol precipitation. Analyze on a 2% agarose gel. The above digestion procedure will primarily yield mononucleosomes. To obtain other fragment lengths, vary the MNase amounts and digestion time. 29. Continue to the ChIP experiment (Step 30). Chromatin Immunoprecipitation (ChIP)
Do NOT block beads with any nonspecific DNA or RNA.
30. Place 40 µL of Dynabeads Protein A into each of two microcentrifuge tubes. Wash each with 600 µL of 1X PBS. Place the tubes in a magnetic stand and aspirate off the PBS. Add 100 µL of 1X PBS to each tube. Add 4 µg of antibody to one tube and 4 µg of preimmune serum to the other. Rotate for 4-6 h at 4°C. 31. Using the magnetic stand, aspirate the supernatants and wash the beads in each tube twice with 0.2 mL of 1X PBS to remove free IgGs (5 min each wash). 32. Remove the supernatant from the beads. Add 500 µL of chromatin extract (from 107 cells) to the beads in each tube. Rotate at 4°C overnight. 33. Wash the beads (10 min each wash): i. Twice with 1 mL of RIPA buffer ii. Twice with 1 mL of RIPA buffer + 0.3 M NaCl iii. Twice with 1 mL of LiCl buffer iv. Once with 1 mL of 1X TE + 0.2% Triton X-100 v. Once with 1 mL of 1X TE For less stringent washing, reduce the concentration of NaCl added to the RIPA buffer and/or perform only a single wash at each step (Steps 33.i-33.v). 34. Resuspend the beads in 100 µL of 1X TE per tube. Add 3 µL of 10% SDS and 5 µL of 20 mg/mL proteinase K to each tube. Incubate overnight at 65°C. 35. Extract and precipitate the DNA as follows: i. Vortex briefly and transfer the supernatants to new tubes using a magnetic stand. ii. Wash the beads with 100 µL of 1X TE and 0.5 M NaCl. Combine each wash solution with the corresponding supernatant. iii. Extract each tube once with 200 µL of phenol:chloroform. iv. Add 2 µL of 20 mg/mL glycogen, 20 µL of 3 M NaOAc (pH 5.3), and 500 µL of ethanol to each tube to precipitate the DNA. 36. Wash each pellet once with 70% ethanol. 37. Resuspend each 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. Enrichment of the region of interest should be significantly higher in the ChIP sample obtained using antibodies than in the sample prepared using preimmune serum. | For 25 µL final volume | For 20 µL final volume |
| | 2X 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 | | H2O | 5.5 µL | 4 µL | | DNA template | 2 µL | 2 µL |
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DNA Repair and Solexa Library Construction
39. Repair DNA ends to generate blunt-ended DNA using the DNA End-Repair Kit (Epicentre). Combine: 1-34 µL of DNA (0.3 µg) 5 µL of 10X End-Repair Buffer 5 µL of 2.5 mM each dNTP 5 µL of 10 mM ATP H2O to adjust the reaction volume to 49 µL 1 µL of End-Repair Enzyme Mix (T4 DNA Pol + T4 PNK) Incubate at room temperature for 45 min. 40. Use MinElute PCR Purification Kit or phenol:chloroform extraction to purify DNA. Elute or resuspend DNA in 30 µL of 1X TE, pH 7.4. 41. Add \"A\" to 3 ends. i. Combine: 30 µL of DNA from Step 40 2 µL of H2O 5 µL of 10X Taq buffer 10 µL of 1 mM dATP 3 µL of Taq DNA polymerase (5 U/µL) Incubate at 70°C for 30 min. ii. Purify using MinElute PCR Purification Kit or phenol:chloroform extraction. iii. Elute with or resuspend in 10 µL of 1X TE, pH 7.4. Alternatively, Klenow fragment exo- can be used for adding \"A\" bases. 42. Perform linker ligation. Combine: 10 µL of DNA (300 ng) 9.9 µL of H2O 2.5 µL of T4 DNA ligase buffer (10X) 0.1 µL of Adapter oligo mix 2.5 µL of T4 DNA ligase (400 U/µL) Incubate at 20°C-23°C for 30 min and then at 16°C overnight. 43. Purify DNA using MinElute PCR Purification Kit. Elute with 20-25 µL of buffer EB. 44. Perform size selection: i. Load 20 µL of linker-ligated DNA onto a 2% E-Gel and perform electrophoresis. ii. Cut the gel around the 200-400 bp region. DNA is not visible on the gel at this step. Load only one sample per gel to avoid contamination. iii. Extract the DNA using MinElute Gel Extraction Kit. Elute with ~11.5 µL of buffer EB. 45. Amplify the DNA using Illumina PCR primers PE 1.0 and PE 2.0 and 2X Phusion HF Master Mix. i. Combine: 10.5 µL of DNA 12.5 µL of 2X Phusion HF Master Mix 1 µL of PCR primer PE 1.0 (2X diluted with 1X TE, pH 7.4) 1 µL of PCR primer PE 2.0 (2X diluted with 1X TE, pH 7.4) ii. Denature at 98°C for 30 sec, followed by 18 amplification cycles (98°C, 10 sec; 65°C, 30 sec; 72°C, 30 sec.) iii. Check 2.5 µL of product on a 2% agarose gel. If the band is not clearly visible, perform three more cycles and check again. 46. Purify the amplified products: i. Load the amplified DNA on a 2% agarose gel and perform electrophoresis. ii. Excise the band at the 200-400 bp region. iii. Purify the DNA using a MinElute Gel Extraction Kit and elute in 20 µL of buffer EB. iv. Measure the DNA concentration using a spectrometer. The DNA can be used for cluster generation using Illumina flow cells and sequencing-by-synthesis on an Illumina Genome Analyzer, following manufacturer’s instructions. ACKNOWLEDGMENTS
The work in the authors’ laboratory was supported by the Intramural Research Program of National Heart, Lung and Blood Institute, National Institutes of Health.
