title: GSM3453041: H3K27me3_mut_rep1_input_Lib_K10E_rec; Mus musculus; ChIP-Seq
library_construction_protocol: Cells were trypsinised, washed in PBS and counted. For SmcHD1 and Rad21 5x107 cells were then crosslinked in 2M Ethylene glycol-bis(succinic acid N-hydroxysuccinimide ester) (EGS, Sigma) in PBS at RT for 1hr followed by 15 min crosslinking in 1% formaldehyde. For H3K27me3 and CTCF 1x107 cells were crosslinked in 1% formaldehyde alone for 15 min at RT. Formaldehyde was quenched by addition of glycine to a final concentration of 125mM and incubation at RT for 3min. Cells were spun down at 700rpm for 4 min at 4oC and lysed in 50mM HEPES pH 7.9, 140mM NaCl, 1mM EDTA, 10% glycerol, 0.25% Triton X100 and 2% NP40. Cellular lysis was assisted by 20 strokes of large clearance pestle in Dounce grinder and subsequent incubation for 10 min at 4oC with constant rotation. Released nuclei were spun down and washed once in 10mM Tris-HCl pH8.0, 200mM NaCl, 1mM EDTA and 0.5mM EGTA. Nuclei were lysed by addition of 0.1% sodium deoxycholate and 0.5% N-lauroylsarcosine. All solutions contained cOmplete EDTA-free protease inhibitors (Sigma) added fresh. Chromatin was sonicated on BioRuptor sonicator (Diagenode) to produce fragments of approximately 500 bp. Triton X100 was added to a final concentration of 1%, and insoluble pellet was removed by centrifugation at maximum speed for 10 min at 40C. Chromatin was aliquoted and stored at -80C until use.  Immunoprecipitation was performed overnight at 40C with 3-5g of specific antibody and 100 l of chromatin corresponding to 1x105 (H3K27me3, CTCF) or 5x105 cells (Rad21, SmcHD1) in 20 mM Tris-HCl pH 8.0, 1mM EDTA, 150 mM NaCl and 1% triton-x-100, with proteinase inhibitors. Antibody-bound chromatin was isolated on rProtein A Sepharose Fast Flow beads (GE Healthcare) that had been blocked for 1 h at 4°C with 1 mg/ml bovine serum albumin (NEB) and 1 mg/ml yeast tRNA (Sigma). Agarose beads with immunoprecipitated material were washed thoroughly in low salt buffer (LSB, 20mM Tri-HCl pH8.0, 2mM EDTA, 150mM NaCl, 0.1% SDS, 1% Triton X100), high salt buffer (HSB, the same as LSB but with 500mM NaCl), LiCl buffer (10MM Tris-HCl pH8.0, 1mM EDTA, 0.25 M LiCl, 1% NP40, 1% deoxycholate) and twice in TE buffer, all with proteinase inhibitors. Immunoprecipitated material was eluted from beads in elution buffer (0.1M NaHCO3, 1% SDS) with shaking on Thermomixer at RT for 30min.  Beads were removed by centrifugation, eluted chromatin was reverse cross-linked and treated with RNAse and proteinase K in the presence of 200mM of NaCl at 370C for 2 hrs followed by 650C overnight with shaking at 800rpm for 1 min every 2 min. DNA was purified using ChIP DNA Clean and Concentrator kit (Zymo Research) according to the manufacturer's instructions.  DNA size was assessed on Bioanalyser (Agilent) and DNA was post-sonicated on Bioruptor Pico sonicator for 18-20 cycles of 30s on/30s off if necessary. DNA Concentration was quantified using PicoGreen® dsDNA Quantitation Kit (Molecular Probes), Bioanalyser High sensitivity DNA assay and/or Qubit dsDNA HS assay kit. NEBNext (H3K27me3) or NEBNext Ultra II (CTCF, Rad21, SmcHD1) DNA Library Prep Kits for Illumina were used to prepare libraries for sequencing, following the manufacturer's instructions.
attributes.tag: GEO Accession
attributes.value: GSM3453041