Mouse Embryonic Fibroblasts 4 days post SLHR induction
antibody
NA
Sequenced DNA Library
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
10 million cells were crosslinked using 1% formaldehyde for 10 min at room temperature followed by quenching by using 200 mM glycine. After that, cell were lysed using a lysis buffer consisting of 10 mM Tris-Cl (pH 8), 100 mM NaCl, 10 mM EDTA, 0.25% Triton X-100 and protease inhibitor cocktail (Roche). Cells were then resuspended in 1% SDS lysis buffer composed of 50 mM HEPES-KOH (pH 7.5), 150 mM NaCl, 1% SDS, 2 mM EDTA, 1% Triton X-100, 0.1% NaDOC and protease inhibitor cocktail. The suspension was nutated for 15 mins at cold room before spinning down to collect the chromatin pellet. The pellet was then washed two times with 0.1% SDS lysis buffer containing 50 mM HEPES-KOH (pH 7.5), 150 mM NaCl, 0.1% SDS, 2 mM EDTA, 1% Triton X-100, 0.1% NaDOC, 1 mM PMSF and protease inhibitor cocktail. Chromatin was then fragmented by sonication which was conducted with cells on ice with 11 cycles, power amplitude of 35% and 30 sec pulses on with 59.9 s pulses off (Branson Sonifier 250). The chromatin solution was clarified by centrifugation at 20,000 g at 4°C for 45 mins and then pre-cleared with Dynabeads protein A (Life Technologies) for 2 hrs at 4°C. The pre-cleared chromatin sample was incubated with 50 μl of Dynabeads protein A loaded with 5 µg antibody overnight at 4°C. The beads were washed three times with 0.1% SDS lysis buffer, once with 0.1% SDS lysis buffer/0.35 M NaCl, once with 10 mM Tris-Cl (pH 8), 1 mM EDTA, 0.5% NP40, 0.25 LiCl, 0.5% NaDOC, and once with TE buffer (PH8.0). The immunoprecipitated material was eluted from the beads by heating for 45 min at 68°C in 50 mM TrisCl (pH 7.5), 10 mM EDTA, 1% SDS. Reverse crosslinking was carried out by incubating the samples with 1.5 μg/ml Pronase at 42°C for 2 hr followed by a 6 hours incubation at 67°C. DNA was then purified using MinElute PCR Purification kit (Qiagen).