GSM7646192: Large preB cell, WT, NIPBL, rep1; Mus musculus; ChIP-Seq
ChIP-seq experiments was performed as previously described (Hu et al, Genes Dev. 2016). Briefly, cells were first crosslinked with 2mM DSG for 45 min and then with 1% formaldehyde for 10 min at room temperature. Cells were then lysed and sonicated in RIPA buffer containing 0.2% SDS (50mM Hepes pH 7.9, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% Na-deoxycholate, 0.2% SDS, 0.5 mM PMSF, and 1x Protease inhibitor cocktail (Roche). Chromatin was cleared by centrifugation at 18,000xg for 20 minutes, and then incubated with 5 μg of antibodies pre-bound to Dynabeads protein G (Life technologies). After extensive washing on beads, bound chromatin was eluted and de-crosslinked in elution buffer (50 mM Tris pH8, 10 mM EDTA, 1% SDS, 0.3M NaCl) overnight at 65°C and purified using the ChIP DNA Clean & Concentrator kit (Zymo Research). Purified ChIP DNA was end repaired, end adenylated, and then ligated with Illumina Truseq indexed adaptors. The ligated DNA was purified with AMPure XP beads (Beckman Coulter) and then amplified with KAPA HiFi DNA Polymerase (KAPA Biosystems) for 8 to 13 cycles. After amplification, the library DNA was size selected with AMPurex XP beads to 200-600 bp range, and the purified libraries were multiplexed for sequencing in house with an Illumina NextSeq 550 system.