title: GSM5343125: shCont_TSC_H3K9me3_rep2; Mus musculus; ChIP-Seq
library_construction_protocol: ChIP were performed as described previously (Harada et al., 2012). Briefly, 1x107 ESCs or TSCs were collected by trypsinization, and fixed using truChIP Chromatin Shearing Kits (Covaris #520154). Totally 2x107 cells were suspended with 2 ml of ChIP buffer (10 mM Tris-HCL pH8.0, 200 mM KCL, 1 mM CaCL2, 0.5%NP40, protease inhibitor cocktail) and incubated on ice for 5 min. Cells were sonicated using Handy Sonic (Tomy Seiko #UR-20P) with 5 sec sonication (intensity 7) and 15 sec interval for three times, then incubated with micrococcal nuclease (NEB #M0247) for 25 min at 37℃. Reaction was stopped by EDTA, then lysates were centrifuged by 15,000 rpm for 15 min at 4°C. Aliquot of supernatants were kept for input. Antibodies were pre-bounded to Dynabeads Protein G or Protein A (Invitrogen #10001D and #10004D) for at least 2 hours at 4°C with rotation. Supernatants were incubated with beads complexes at 4°C with rotation, then the immunocomplexes were washed by using ChIP buffer for 2 times, wash buffer (10 mM Tris-HCL pH8.0, 500 mM KCL, 1 mM CaCL2, 0.5%NP40, protease inhibitor cocktail) for 2 times, and TE buffer for one time. These complexes were eluted by elution buffer (10 mM Tris-HCl, 1% SDS, 5 mM EDTA and 300 mM NaCl) at 65°C for 30 min. Crosslinking was reversed by incubation at 65°C for 6 h, after which RNAs and proteins were degraded by adding RNase (Ambion #AM2286) at 37°C for 30 min and Proteinase K (Zymo Research #D3001-2) at 55°C for 2 hours, respectively. ChIPed-DNAs were purified using QIAquick PCR purification kits (Qiagen #28106). ChIP-seq libraries were prepared using NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB #E7645) according to manufactures instructions. Single-end 65 bp sequencing was performed on a Hiseq 2500.
attributes.tag: GEO Accession
attributes.value: GSM5343125