GSM8796319: SMARCA5 Mnase Chip-seq in SMARCA5 KOBMDM, untreated, replicate1; Mus musculus; ChIP-Seq
For ChIP-seq, nuclear extracts were prepared as described in Gualdrini et al., 2022. Chromatin was sheared by sonication (Branson Ultrasonics Sonifier or Diagenode Bioruptor Pico). For SMARCA5 MNase ChIP-seq, nuclei were digested with MNase (9 U; Roche) in digestion buffer (20 mM Tris-HCl, pH 7.6; 5 mM CaCl₂) at 37°C for 2 hours, followed by EDTA quenching (50 mM). Digested chromatin was sonicated in lysis buffer (10 mM Tris-HCl, pH 8.0; 100 mM NaCl; 1 mM EDTA; 0.5 mM EGTA; 0.1% sodium deoxycholate; 0.5% sodium lauroyl sarcosinate) to promote nuclear lysis. Immunoprecipitations were performed with target-specific antibodies prebound to Dynabeads™ Protein G. Beads were washed with RIPA buffer and TE buffer, and DNA was eluted, de-crosslinked, and purified using Qiagen kits (QIAquick PCR purification or MinElute PCR purification). DNA libraries were prepared using the NEBNext Ultra II DNA Library Prep Kit (NEB) or following Gualdrini et al., 2022, protocols. Libraries were assessed for size and quality using the Agilent TapeStation High Sensitivity D5000 assay. Libraries were diluted to 4 nM and sequenced as paired-end reads (51-bp or 61-bp) on an Illumina NextSeq6000 platform.