Name: GSM7123809
Instrument: NextSeq 2000
Strategy: ChIP-Seq
Source: GENOMIC
Selection: ChIP
Layout: SINGLE
Construction protocol: Cells were cross linked in 3 mM disuccinimidyl glutarate for 30 min then in 1% formaldehyde for 10 min at room temperature. After quenching with 125 mM glycine, the cells were washed in 1× PBS, pelleted, flash frozen in liquid nitrogen, and stored at 80 °C. Cell pellets were thawed on ice and incubated in lysis solution (50 mM Hepes KOH pH 8, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP-40, and 0.25% Triton X-100) for 10 min. The isolated nuclei were washed (10 mM TrisHCl pH 8, 1 mM EDTA, 0.5 mM EGTA, and 200 mM NaCl) and sheared in (0.1% SDS, 1 mM EDTA, and 10 mM TrisHCl pH 8) with the Covaris E229 sonicator for 10 min. After centrifugation, chromatin was immunoprecipitated overnight at 4 °C with 1:50 T-bet/TBX21 (Cell Signaling Technology, mAb #97135S). The next day, the antibody-bound DNA was incubated with Protein A+G Dynabeads (Invitrogen) in ChIP buffer (50 mM Hepes KOH pH 7.5, 300 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, and 0.1% SDS), washed, and treated with Proteinase K and RNase A and reverse cross linked. Purified ChIP DNA was used for library generation (NEBNext Ultra II DNA Library Prep Kit for Illumina) according to manufacturer's instructions