GSM5739003: H3K9me3_control; Homo sapiens; ChIP-Seq
TGF-β1 stimulated cells were treated with Formaldehyde to a final concentration of 1% to crosslink Proteins to DNA for 10 minutes at room temperature. Glycine was added at a final concentration of 125mM to quench the formaldehyde. Cells were washed twice with Phosphate Buffered Saline, scraped and centrifuged. The cell pellet was resuspended in ChIP lysis buffer (50 mM HEPES-KOH pH7.5, 140 mM NaCl, 1 mM EDTA pH8, 1% Triton X-100, 0.1% Sodium Deoxycholate, 0.1% SDS and Protease Inhibitors). Cell were lysed and sonicated using Diagenode Bioruptor Pico under 30 seconds ON and 30 seconds OFF for 30 cycles condition to generate DNA-Protein Complex fragments. 50uL of chromatin was removed to serve as input sample and rest of the chromatin was immunoprecipitated using ChIP-grade H3K9me3 Antibody (Santa Cruz, Cat# sc-130356). The immunoprecipitated chromatin was purified using Protein G Sepharose beads and washed once in low salt wash buffer, once in high salt wash buffer and once in LiCl wash buffer. DNA was eluted using elution buffer and treated with 5M NaCl to reverse cross-link followed by treatment with RNaseA (Invitrogen, Cat# 12091021) and Proteinase K (Sigma-Aldrich, Cat#P2308). DNA was purified using PCR Purification kit (Himedia, Cat# MB512) and was stored at -20℃ until further use. Libraries for ChIP sequencing were prepared using NEBNext® Ultra II DNA Library preparation kit (Cat# E7645). In brief, ChIP DNA and the Input DNA were subjected to various enzymatic steps for repair the ends and tailing with dA-tail followed by ligation of adapter sequences. These adapter ligated fragments were then size selected using SPRI beads. Next, the size selected fragments were indexed during limited cycle PCR to generate final libraries for paired-end sequencing.