title: GSM5733521: 170kb Enhancers + SynHoxA assemblon in HoxA -/- mESCs, 0h of RA+SAG differentiation, replicate 1, H3K27Ac ChIP; Mus musculus; ChIP-Seq
library_construction_protocol: Cells were collected at 0h or 24h, 48h, or 96h after RA/SAG treatment. Cells were crosslinked at room temperature in 1 mM DSG (ProteoChem) for 15 min, followed by the addition of 1% FA (vol/vol) for 15 min. The reaction was quenched with Glycine and cells were washed with 1 × PBS. Samples were divided into ~25-30x106 cell aliquots, pelleted by centrifugation at 275 g, and frozen at -80°C. Cell aliquots were thawed on ice and lysis was performed in 5 mL 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 pelleted by centrifugation for 5 min at 1200 g, resuspended in 5 mL 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 5 min at 1200 g and resuspended in 2 mL 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). For sonication, each sample was split in two Bioruptor tubes with added sonication beads. Sonication was performed using the Bioruptor Pico (Diagenode) for 18 cycles of 30 sec on and 30 sec off to sheer DNA into an average size of approximately 200bp. Immunoprecipitation was performed for 16h at 4°C on a rotating platform by incubating with Dynabeads protein-G (Thermo Fisher Scientific) conjugated with antibodies. For histone modifications, half of each original cell aliquot was incubated with Dynabeads protein-G conjugated with 5 µg of rabbit polyclonal to Histone H3K27me3 (Active motif 39155) or rabbit polyclonal to Histone H3 (acetyl K27) (Abcam ab4729) antibodies. For CTCF, the entire cell aliquot was incubated with Dynabeads protein-G conjugated with 5 µl of rabbit polyclonal to CTCF (EMD 07-729) antibody. After the immunoprecipitation, washes were performed with the following ice-cold buffers: sonication buffer, sonication buffer with 500 mM NaCl, LiCl wash buffer (20 mM Tris-HCl pH 8.0, 1 mM EDTA pH 8.0, 250 mM LiCl, 0.5% Igepal (vol/vol), 0.5% sodium deoxycholate (wt/vol)), and TE buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA pH 8). Elution was performed in elution buffer (50 mM Tris-HCl pH 8.0, 10 mM EDTA pH 8.0, 1% SDS (vol/vol)) by incubating for 45 min at 65°C with occasional flicking of the tube. Samples were incubating for 16h at 65°C to perform reversal of crosslinks. 200 μL of TE and RNase A (Sigma) at a final concentration of 0.2 mg/mL was added to digest RNA by and incubating for 2h at 37°C. Proteins were digested by adding Proteinase K (Invitrogen) at a final concentration of 0.2 mg/mL, supplemented with CaCl2, at 55°C for 30 min. DNA was purified with phenol:chloroform:isoamyl alcohol (25:24:1; vol/vol) (Invitrogen) followed by an ethanol precipitation. DNA pellets were resuspended in 70 μL of water. lllumina DNA sequencing libraries were prepared with approximately one third of the ChIP sample (24 μL) 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. Agencourt AmpureXP beads (Beckman Coulter) were used to remove unligated adapters. PCR amplification was performed with Phusion polymerase (New England Biolabs) and TruSeq primers (Sigma). Libraries were gel purified (Qiagen) between 250 and 550bp in size. Libraries were quantified before pooling using the KAPA library amplification kit on the Roche Lightcycler 480 or the Bio-Rad CFX96. The libraries were sequenced on Illumina NextSeq 500 using V2.5 chemistry (75 cycles, single-end 75bp) or on Illumina NovaSeq 6000 using the SP Reagent Kit (100 cycles, single-end 100bp) at the Genomics Core Facility at NYU.