title: GSM3136836: ChIPseq A[N]bHLH.HA in EB+12hrDox(iAscl1[bHLH:Ngn2].HA) replicate 1; Mus musculus; ChIP-Seq
library_construction_protocol: ChIP-seq: Cells were collected at 12 hours and 48 hours after TF induction and fixed with 1mM DSG (ProtoChem) followed by 1% FA (vol/vol) each for 15 min at room temperature. Pellets containing 25-30x10^6 cells were aliquoted and flash-frozen at -80°C. Cell aliquots were thawed on ice and resuspended in 5 ml of cold lysis buffer 1 (50 mM HEPES-KOH pH 7.5, 140 mM NaCl, 1 mM EDTA, 10% glycerol (vol/vol), 0.5% Igepal (vol/vol), 0.25% Triton X-100 (vol/vol)) with 1X protease inhibitors (Roche, 11697498001) and incubated at 4°C on a rotator. The samples were centrifuged for 5 min at 2300 rpm at 4°C and resuspended in 5ml of cold lysis buffer 2 (50 mM HEPES-KOH pH 7.5, 140 mM NaCl, 1 mM EDTA, 10% glycerol (vol/vol), 0.5% Igepal (vol/vol), 0.25% Triton X-100 (vol/vol)) with 1X protease inhibitors and incubated 10 min at 4°C on a rotator. Nuclear extracts were centrifuged for 5 min at 2300 rpm resuspended in cold 3 ml of sonication buffer (50mM HEPES pH 7.5, 140 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 0.1% sodium deoxycholate (wt/vol), 0.1% SDS (wt/vol). Sonication of the nuclear extracts was performed on ice with Branson 450 digital sonifier (Marshall Scientific, B450CC) at 20% amplitude, 18 cycles of 30s ON/60s OFF, to sheer crosslinked chromatin into average size of approximately 300 bp. Immunoprecipitation of the sonicated chromatin with Dynabeads protein-G (Thermo Fisher) conjugated antibodies were done overnight (16 hr) at 4°C on a rotator. 5 ug of following antibodies were used for immunoprecipitation. In-house library preparation kit was used for ChIP-seq: 24ul of ChIP DNA (1:100 dilution of input DNA) was used to prepare lllumina DNA sequencing libraries. Bioo Scientific multiplexed adapters were ligated after end repair and A-tailing, and unligated adapters were removed by purification using Agencourt AmpureXP beads (Beckman Coulter). Adapter-ligated DNA was amplified by PCR using TruSeq primers (Sigma). DNA libraries between 300 and 500 bp in size were purified from agarose gel purified using Qiagen minElute columns. and the final quantification of the library before pooling was done with KAPA library amplification kit (Roche Lightcycler 480).
attributes.tag: GEO Accession
attributes.value: GSM3136836