GSM3272840: MEF_Input_ChIP; Mus musculus; ChIP-Seq
5-10x106 cells were cross-linked with 1% paraformaldehyde for 10 min and stopped by adding Glycine (to be 125 mM). The cross-linked material was washed with buffer 1 (0.25% Triton, 10 mM EDTA, 0.5 mM EGTA, 10 mM Tris pH7.5) and Buffer 2 (200 mM NaCl, 10 mM EDTA, 0.5 mM EGTA, 10 mM Tris pH7.5) supplemented with protease inhibitor cocktail (Roche). Cell pellet was lysed in SDS lysis buffer (50 mM Tris pH 8.0, 10mM EDTA, 1% SDS, 50 mM PMSF) and was sonicated to generate 200 to 600 bp fragments. Fragmented chromatin was immunoprecipitated with magnetic beads coupled with 5 μg of antibody. The beads were washed with low salt buffer (50 mM Tris pH 8.0, 150 mM NaCl, 0.1% SDS, 0.5% Deoxycholate, 1% NP40, 1 mM EDTA), high salt buffer (50 mM Tris pH 8.0, 500 mM NaCl, 0.1% SDS, 0.5% Deoxycholate, 1% NP40, 1 mM EDTA), LiCl wash buffer (50 mM Tris pH 8.0, 250 mM LiCl, 0.5% Deoxycholate, 1% NP40, 1 mM EDTA), Morohashi RIPA buffer (50mM Tris pH7.5, 150mM NaCl, 5mM EDTA, 0.5% NP40, 0.1% SDS), DOC/Triton Buffer (25mM Tris pH7.5, 150mM NaCl, 5mM EDTA, 1% Triton-X-100, 0.5% DOC) and Tris-EDTA buffer (10 mM Tris pH 8.0, 1 mM EDTA) for 10 min at 4 °C. Complexes were eluted in elution buffer (1% SDS, 0.1 M NaHCO3) for 2 h at 65 °C and reverse cross-linked for 6 h at 65°C. Reverse cross-linked DNA was purified by QIAquick PCR purification kit ChIP DNA samples for sequencing were prepared as described for ChIP above. PCR cleanup with MinElute PCR Purification kits (Qiagen) yielded 20 μl  ChIP DNA which was used immediately in a 23.3 μl combined end repair and A-tailing reaction using a KAPA Hyper Prep Kit (Kapa Biosciences) for 30 minutes at 20°C followed by 30 minutes at 65°C. 10 μl of ligase buffer, 3.7 μl of Y-Adapters diluted 1:50 (Illumina 5179206) and 3.3 μl ligase (KAPA Hyper Prep Kit) were added and incubated at 20°C for 4 hours. Double-stranded DNA fragments were purified from this reaction using KAPA Pure Beads (Kapa Biosciences) and eluted in 22 μl 10 mM Tris pH8.0. The amplification cycle number was determined empirically by using 1 μl  of the eluent in a 10 μl  test reaction containing a 1:10,000 dilution of SYBR Green I (Invitrogen), 2x KAPA HotStart ReadyMix (Kapa KM2618  and custom barcoding primers. An appropriate number of cycles was used in a subsequent 50 μl reaction with the remaining eluent in the absence of SYBR Green. Libraries were size-selected on a 2% E-Gel EX agarose gel (Invitrogen) and fragments between 150 and 350 bp were extracted using a QIAEX II Gel Extraction Kit (Qiagen) performed at room temperature. Libraries were quantitated with a high sensitivity Qubit dsDNA HS Assay Kit (Invitrogen Q32854) and pooled prior to submission quality control analyses and next-generation sequencing.