title: GSM3240325: H3K27Ac on A673 STAG2 null; Homo sapiens; ChIP-Seq
library_construction_protocol: Chromatin immunoprecipitation sequencing for EWS/FLI, H3K27Ac, and CTCF: Chromatin was sheared to about 200 bp fragments using the Covaris ultra-sonication. The lysate was diluted with ChIP dilution buffer (16.7 mM Tris-HCl pH 8.1, 16.7 mM NaCl, 1.2 mM EDTA, 1.1% Triton-X100, 0.01% SDS) and cleared by centrifugation. 5% of the supernatant volume set aside as an input control. The remaining lysate was incubated over night at 4°C with 5 ug of antibody (3 ug for histone antibodies). The following antibodies were used for ChIP: Fli (sc-356X, Santa Cruz Biotechnology), H3K27Ac (ab4729, Abcam), and CTCF (07-729, EMD Millipore).   Chromatin immunoprecipitation sequencing for SMC1a: Cells were prepared for ChIP as follows. All buffers contained freshly prepared 1× cOmplete protease inhibitors (Roche, 11873580001). Frozen crosslinked cells were thawed on ice and then resuspended in lysis buffer 1 (50 mM HEPES-KOH, pH 7.5, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP-40, 0.25% Triton X-100, 1× protease inhibitors) and rotated for 10 minutes at 4°C, then spun at 1350 rcf for 5 minutes at 4°C. The pellet was resuspended in lysis buffer 2 (10 mM Tris-HCl, pH 8.0, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 1× protease inhibitors) and rotated for 10 minutes at 4°C and spun at 1350 rcf for 5 minutes at 4°C. The pellet was resuspended in sonication buffer (20 mM Tris-HCl pH 8.0, 150 mM NaCl, 2 mM EDTA pH 8.0, 0.1% SDS, and 1% Triton X-100, 1× protease inhibitors) and then sonicated on a Misonix 3000 sonicator for 10 cycles at 30 seconds each on ice (18-21 W) with 60 seconds on ice between cycles. Sonicated lysates were cleared once by centrifugation at 16,000 rcf for 5 minutes at 4°C. 50 µL was reserved for input, and then the remainder was incubated overnight at 4°C with magnetic beads bound with antibody to enrich for DNA fragments bound by the indicated factor. Chromatin immunoprecipitation sequencing for EWS/FLI, H3K27Ac, and CTCF: The precipitated complexes were bound to protein A/G high-capacity agarose beads (Pierce) for 2 h before washing sequentially with ice cold low salt wash (20 mM Tris-HCl pH 8.1, 150 mM NaCl, 2 mM EDTA, 1% Triton-X100, 0.1% SDS), high salt wash (20 mM Tris-HCl pH 8.1, 2 mM EDTA, 500 mM NaCl, 1% Triton-X100, 0.1% SDS), LiCl wash (10 mM Tris-HCl pH 8.1, 0.25 M LiCl, 1 mM EDTA, 1% deoxycholic acid, 1% IGEPAL CA-630) and TE wash (10 mM Tris-HCl pH 8.1, 1 mM EDTA) and eluted in elution buffer (0.1 M NaHCO3, 1% SDS). Library preparation for eluted DNA fragments were performed using the NEBNext ChIP-Seq Library Prep for Illumina (New England Biolabs). The Pippin Prep (Sage Science) was used to size select the adaptor ligated DNA in the 175-450 bp range prior to PCR enrichment. Libraries were sequenced on the Illumina HiSeq 2000 instrument.  Chromatin immunoprecipitation sequencing for SMC1a: For ChIP-seq experiments, purified ChIP DNA was used to prepare Illumina multiplexed sequencing libraries.