title: GSM4773698: ChIPseq Hoxc13:c10.1 in EB-Day3[D0+RA+SAG][D2+24hDox(iFlag.Hoxc13:c10.1(399))] replicate 2; Mus musculus; ChIP-Seq
library_construction_protocol: Cells were collected 24h and 48h after Doxycycline (Dox) treatment. Crosslinking was performed at room temperature in 1mM DSG (ProteoChem) for 15min, followed by the addition of 1% FA (vol/vol) for an additional 15min. After quenching with Glycine, cells were divided into ~25-30x106 aliquots, pelleted by centrifugation and frozen at -80°C. After thawing cells on ice, lysis was performed in 5mL of 50 mM HEPES-KOH pH 7.5, 140 mM NaCl, 1 mM EDTA pH 8.0, 10% glycerol (vol/vol), 0.5% Igepal (vol/vol), 0.25% Triton X-100 (vol/vol) with 1× protease inhibitors (Roche, 11697498001) for 10 min at 4 °C. Cells were centrifuged for 5min, resuspended in 5mL of 10 mM Tris-HCl pH 8.0, 200 mM NaCl, 1 mM EDTA pH 8.0, 0.5 mM EGTA pH 8.0 with 1× protease inhibitors, and incubated for 10 min at 4 °C on a rotating platform. Cells were centrifuged for 5min and resuspended in 2mL of Sonication Buffer (50 mM Hepes pH 7.5, 140 mM NaCl, 1 mM EDTA pH 8.0, 1 mM EGTA pH 8.0, 1% Triton X-100 (vol/vol), 0.1% sodium deoxycholate (wt/vol), 0.1% SDS (vol/vol) with 1× protease inhibitors). Sonication was performed by splitting each sample to 2 Bioruptor tubes with sonication beads and using the Bioruptor Pico (Diagenode) for 18 cycles of 30 sec on and 30 sec off into an average size of approximately 200 bp. Immunoprecipitation was performed for 16h at 4 °C on a rotating platform by incubating with Dynabeads protein-G (Thermo Fisher Scientific) conjugated with 5 µg of one of the following antibodies: mouse monoclonal antibody to Flag (Sigma, F1804) or rabbit polyclonal antibody to HA (Abcam, ab9110). lllumina DNA sequencing libraries were prepared with one third of the ChIP sample or a 1:100 dilution of the input sample in water. Library preparation was performed by end repair, A-tailing and ligating Illumina-compatible Bioo Scientific multiplexed adapters. Unligated adapters were removed using Agencourt AmpureXP beads (Beckman Coulter). Amplification was performed by PCR with Phusion polymerase (New England Biolabs) and TruSeq primers (Sigma). Libraries were gel purified (Qiagen) between 250 and 550bp in size. Final quantification of the library was performed using the KAPA library amplification kit on the Roche Lightcycler 480 before pooling.