ChIP-seq libraries were prepared as previously described46 (link). Briefly, 10 million CD8+ T cells were fixed with 1% formaldehyde at 37 °C for 10 min, sonicated, and fragmented chromatin was immunoprecipitated with anti-STAT5B or anti-H3K27ac antibodies that were coupled to Magna ChIP protein A/G magnetic beads (EMD Millipore, 16-663). ChIP-seq DNA libraries were prepared with the KAPA LTP Library Preparation Kit and barcoded with NEXTflex DNA barcodes, quantified, and sequenced on an Illumina HiSeq 3000 system.
Protocol detail
CD8+ T Cell Chromatin Immunoprecipitation
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Corresponding Organization :
Other organizations : National Heart Lung and Blood Institute, National Institutes of Health, National Cancer Institute, Johns Hopkins Medicine, Johns Hopkins University, Stanford University, National Institute of Diabetes and Digestive and Kidney Diseases, National Institute of Allergy and Infectious Diseases, Center for Cancer Research, Howard Hughes Medical Institute
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Variable analysis
independent variables
- Anti-STAT5B antibody
- Anti-H3K27ac antibody
- Chromatin immunoprecipitation (ChIP) of STAT5B and H3K27ac binding sites
- CD8+ T cells
- 1% formaldehyde fixation at 37 °C for 10 min
- Sonication to fragment chromatin
- Magna ChIP protein A/G magnetic beads
- KAPA LTP Library Preparation Kit
- NEXTflex DNA barcodes
- Illumina HiSeq 3000 sequencing
- Positive control: Not explicitly mentioned
- Negative control: Not explicitly mentioned
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