GSM7152452: H3K4me3_CD34_2; Homo sapiens; ChIP-Seq
Approximately 2.5 × 10^7 cells were used for each immunoprecipitation. Cells were cross-linked with 1% formaldehyde for 10 min at room temperature with rotation, and the reaction was quenched with glycine at a final concentration of 125 mM. Cross-linked cells were then lysed and resuspended in 2 mL of RIPA buffer and sonicated for 12 cycles with a Branson 250 sonifier (10 s on/90 s off for a total of 2 min of pulses with 20% output from the micro-tip) to obtain fragments of chromatin approximately 200–300 bp in size. Supernatants were precleared by incubation with 200 µL of protein A/G agarose bead slurry (Thermo Fisher Scientific, cat. #15918014) overnight at 4°C with rotation. Meanwhile, 12.5 µg of IP antibody was incubated with 50 µL of protein A/G agarose bead slurry in 1 mL of PBS overnight at 4°C with rotation. Saved precleared chromatin (20 µL) was used as the input sample. Precleared chromatin was incubated with the antibody–bead complex for 7 h at 4°C with rotation. Cross-linking of DNA was reversed by incubation with RNase A (1 µg/µL), proteinase K (0.2 mg/mL), and 0.25 M NaCl overnight at 65°C. Immunoprecipitated DNA was purified using the Qiagen PCR Extraction Kit and eluted with 20 µL of EB elution buffer. Sequencing libraries were prepared using the NEBNext Ultra II DNA Library Prep Kit (NEB, cat. #E7645) with homemade TruSeq adaptors. Libraries were sequenced using an Illumina HiSeq 4000 system.