GSM8184632: A7r5 cells, siRNALemd3, H3K9me3, ChIP, replicate 1; Rattus norvegicus; ChIP-Seq
Cells were crosslinked with 1% formaldehyde for 10 min at room temperature and quenched by the addition of glycine to a final concentration of 125 mM for 5 min. The fixed cells were resuspended in lysis buffer containing protease inhibitors, then subjected to 30 cycles (30 s on and off) of sonication (Bioruptor, Diagenode) to generate chromatin fragments of ~300 bp in length. Lysates were diluted in buffer (1% Triton X-100, 2 mM EDTA, 150 mM NaCl, and 20 mM Tris-HCl, pH 8.1) containing protease inhibitors. For immunoprecipitation, the diluted chromatin was incubated with normal IgG (control) or indicated antibodies at 4℃ overnight with constant rotation, followed by incubation with 50 μl of 50% (v/v) protein A/G Sepharose beads for 2 h. Beads were successively washed. The pulled-down chromatin complex eluted by TE (1 mM EDTA and 10 mM Tris-HCl, pH 8.0) and input were de-crosslinked at 55℃ for 16 h in elution buffer (1% SDS and 0.1 M NaHCO3). The DNA was purified with the QIAquick PCR Purification Kit (QIAGEN). Libraries for ChIP-seq were prepared using VAHTS Universal Pro DNA Library Prep Kit for Illumina following manufacturers's protocols.