GSM7914441: WT_Spermidine_ZDNA_rep2; Mus musculus; ChIP-Seq
Cells were cross-linked with 1% formaldehyde and then lysed for 10 min on ice in RadioImmunoPrecipitation Assay (RIPA) buffer supplemented with complete protease inhibitor cocktail (Roche). Chromatin was fragmented using Covaris for 15 min (duty cycle 2%, intensity 3, cycle/burst 200) and the sheared material was cleared by a 10-min centrifugation at 13,000 rpm at 4 °C. The cleared material was immunoprecipitated overnight at 4 °C with Pol II Ab conjugated to magnetic Protein-G beads (Life Technologies, Dynabeads), followed by extensive washing of the beads with ice-cold RIPA, high-salt RIPA, LiCl. Chromatin was eluted from the beads, treated with 1µg DNase-free RNase (Roche) for 30 min at 37°C and then treated with Proteinase K (Roche) for 2 hrs at 37°C. Chromatin was reverse cross-linked by incubating at 65°C overnight. DNA from reverse cross-linked material was purified with SPRI beads (Agencourt AMPure XP beads, Beckman Coulter); and then subject to sequential steps of end-repair, A-base addition, adaptor-ligation and PCR amplification. DNA was PCR amplified for 15 cycles. Libraries were size-selected for 200–500-bp fragments with SPRI beads.