GSM9316034: KO-SRT4; Mus musculus; ChIP-Seq
Samples were fixed with 1% paraformaldehyde followed by 0.125M glycine to stop crosslinking. Samples were lysed in freshly made sonication buffer (20mM Hepes, 1% SDS, 0.25 protease inhibitor cocktail (Sigma Aldrich)) and sonicated using the Bioraptor Pico (Diagenode). Fragmented chromatin was precleared using bovine serum albumin (BSA)- blocked protein A/G beads (Invitrogen) and 1.7ug of H3K4me3 antibody (Diagenode) was added. Following overnight incubation, immune complexes were captured by incubation with BSA-blocked protein A/G beads for 1 hour at 4C on a rotating wheel. Beads were washed with wash buffer 1 (2mM EDTA, 20mM tris (pH8), 150mM NaCl, 1 Triton X- 100, and 0.1% SDS), buffer 2 (2 mM EDTA, 20 mM tris (pH 8), 150 mM NaCl, 1% Triton X-100, 0.1% SDS, and 500 mM NaCl), and buffer 3 (1 mM EDTA and 10 mM tris (pH 8)) for 5 mins at 4C on a rotating wheel. Samples were eluted for 20 mins at room temperature in 0.1 M NaHCO3 and 1% SDS. Eluted chromatin was de-crosslinked overnight at 65C using 10mg/ml proteinase K and 5M NaCL. De-crosslinked DNA was purified using MiniElute PCR Purification Kit (QIAGEN) library preparation was performed using the KAPA HyperPrep kit (Roche) according to manufacturer's instructions.