title: GSM3081963: HeLa_G2M_ChIP-Seq_rep2; Homo sapiens; ChIP-Seq
library_construction_protocol: For ChIP-Seq, Cells were lysed using nucleosome lysis buffer (60 mM KCl, 15 mM NaCl, 5 mM MgCl2, 0.1 mM EGTA, 15 mM Tris-HCl pH 7.5, 0.2% NP-40) supplemented with 0.1 mM DTT and PPI. Lysates were then passed through a sucrose cushion (50 mM Tris-HCl pH 7.5, 5 mM MgCl2, 25 mM KCl, 1.2M Sucrose) by centrifugation at 3.5g for 15 minutes at 4 ˚C. The pellet (containing the nuclei) was resuspended in the digestion buffer (50 mM Tris-HCl pH 7.5, 4mM MgCl2, 1mM CaCl2, 0.32 M Sucrose) with 200U micrococcal nuclease (Mnase) (Worthington Biochemical) and incubated for 10 minutes at 37 ˚C. Digestion was stopped by the addition of 1 mM each of EDTA and EGTA. Nuclei were pelleted by centrifugation at 3.5g for 10 minutes at 4 ˚C. Pellet was resuspended in 500 µl solubilization buffer ( 50mM HEPES pH 7.6, 500mM LiCl, 1mM EDTA, 0.7% sodium deoxycholate, 0.1% SDS and 1% NP-40) and rotated at 4 ˚C for 1 hour followed by centrifugation at 12,000g for 10 minutes at 4 ˚C. The pellet was discarded and the supernatant, the nucleosome enriched fraction, was used for immunoprecipitations and immunoblot assays. DNA was extracted from a small aliquot of the nucleosome-enriched fraction using phenol: chloroform: isoamyl alcohol (Thermo Fisher Scientific 15593031) and run on a 2% agarose gel to confirm the expected size of the digested DNA (147 bp and multiples thereof).  For immunoprecipitations, nucleosome enriched fractions were diluted to 1 ml in dilution buffer (0.005% SDS, 0.1% Triton X-100, 1.2 mM EDTA, 1.67 mM Tris HCl pH8.0, 167 mM NaCl ) and incubated with antibodies (2 µg/sample) conjugated to Sepharose A or G beads with rotation at 4 ˚C overnight. Beads were washed with dilution buffer 3x and immunoblots were performed as described above. RNAse-A treatment was performed as described in [REF Ast Paper]. ChIP-Seq Libraries for sequencing were prepared using NEBNext Ultra DNA Library Preparation Kit (NEB E7370).
attributes.tag: GEO Accession
attributes.value: GSM3081963