title: GSM5825682: skeletal muscle satellite cells p300 ChIP-seq KO rep1; Mus musculus; ChIP-Seq
library_construction_protocol: Lysates were clarified from sonicated nuclei and p300-DNA complexes were isolated with antibody. The ChIP experiments were performed as described previously.(Rossi et al., 2019) Briefly, lysates were generated from 4 × 106 cells. Each lysate was immunoprecipitated with 5 µg of anti-p300 (ab8580 from Abcam) and the corresponding control IgG, respectively. Antibodies were pre-conjugated to 25 µl of A/G protein-coupled paramagnetic beads (ThermoFisher) in 0.5% PBS/BSA at 4℃. Beads were then added to the lysate and allowed to undergo overnight incubation. Beads were washed six times with modified RIPA buffer (50 mM HEPES [pH 7.6], 500 mM LiCl, 1 mM EDTA, 1% NP-40, and 0.7% sodium deoxycholate) and once with TE containing 50 mM NaCl. DNA was eluted in solution TE containing 2% SDS and cross-linking was incubated at 65 °C for 2 h in reverse. DNA was then purified by Qiaquick columns (Qiagen). The DNA was used for library construction or qPCR analysis using specific primers (Supplementary Table S1). Libraries were constructed using the ChIP Elution Kit and the DNA Smart ChIP Seq Kit from a low input DNA template (Illumina) sequencing library preparation with 75nt paired ends.