GSM4972151: L1 AMO 36 h - H3K9me3 rep2; Mus musculus; ChIP-Seq
Treated cells were harvested by trypsinization and rinsed twice by PBS. And cells were crosslinked by 1% FMA in 1 ml solution with rotation at RT for 10 min. And crosslinking was stopped by addition of 125 mM glycine. Cells were washed with PBS twice and then treated with 200 μl lysis buffer (50 mM Tris-HCl pH 8.1, 10 mM EDTA, 1% SDS, supplied with 1 mM DTT and 1 mM PMSF) on ice for 10 min. Sonication was performed with 10 s on, 20 s off for 5 cycles with 25% amplification. Cells were spun down at 14,000 rpm at 4℃ for 10 min, and supernatant was collected for immunoprecipitation. For ChIP, 1 μl H3K9me3 antibody was added into the sample and incubated at 4℃ overnight, after washing with Low Salt IP Wash Buffer (25 mM Tris-HCl pH 8.1, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% SDS, 0.1% Na-deoxycholate), High Salt IP Wash Buffer (25 mM Tris-HCl pH 8.1, 500 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% SDS, 0.1% Na-deoxycholate), LiCl IP Wash Buffer (10 mM Tris-HCl pH 8.1, 250 mM LiCl, 1 mM EDTA, 0.5% NP-40, 0.1% Na-deoxycholate) and TE buffer (10 mM Tris-HCl pH 8.1, 1 mM EDTA), ChIPed DNA were eluated twice by IP Elution solution (50 mM Tris-HCl pH 8.1, 1% SDS, 1 mM EDTA and supplied with 100 mM NaHCO3, 250 mM NaCl, 80 μg/ml Proteinase K) at 65℃ for 20 min. After reverse crosslinking at 65℃ for 6-16 h, DNA were extracted by AMPure XP beads (Beckman Coulter). ChIP-seq were prepared by library preparation modules (New England Biolabs)