GSM1163119: H3K4me3_CP_Rep_1; Mus musculus; ChIP-Seq
Chromatin immunoprecipitation of histone modifications were performed according to the Young lab protocol (Lee et al., 2006) with minor modification. Briefly, frozen pellets of cross-linked cells (10 3 106) were thawed in cold 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 3 protease inhibitors) and gently rocked at 4  C for 10 min in 14 ml conical tubes. Cells were pelleted at 1350 x g at 4 C in a clinical centrifuge and resuspended in cold lysis buffer 2 (10 mM Tris-HCl, pH 8.0, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 1 3 protease inhibitors) and gently rocked at 4 C for 10 min in 14 ml conical tubes. Cells were pelleted at 1350 x g at 4C in a table top centrifuge and resuspended in 2 ml cold lysis buffer 3 (10 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% Na-Deoxycholate, 0.5% N-lauroylsarcosine, 1 3 protease inhibitors) and sonicated to 200-600 bp fragments using a Diagenode Bioruptor (3 3 10 min cycles, 30 s ON / 30 s OFF at 4 C). Soni- cated lysates were cleared by pelleting insoluble material at 20,000 x g at 4  C followed by incubation with antibody bound Protein A/G magnetic beads (2.5 mg Ab / 50uL beads / IP) in 1 ml of 0.5% BSA/PBS overnight at 4 C. Magnetic beads were washed 3 times with block (0.5% BSA/PBS), incubated for approximately 4 hr at 4 C with antibody in block and then washed 3 times with block prior to addition of cleared cell lysates. Immunoprecipitated material was washed five times with cold wash buffer (RIPA: 50 mM HEPES-KOH, pKa 7.55, 500 mM LiCl, 1 mM EDTA, 1.0% NP-40, 0.7% Na-Deoxycholate) and one time with TE plus NaCl, followed by elution and uncrosslinking in 210 uL of 1% SDS in TE overnight at 65  C. 200 uL of uncrosslinked material was treated with RNase A for 2 hr, proteinase K for 2 hr and extracted 2 times with phenol chloroform isoamyl alcohol, followed by ethanol precipitation with a glycogen coprecipitant, 80% ethanol wash and final resuspension in TE. Illumina sequencing libraries were generated (Schmidt et al., 2009), with minor modi- fication. Briefly, 5-50 ng of immunoprecipitated nucleic acid was end repaired, a-tailed and ligated to Illumina single end adapters using an NEB Next kit (New England Biolabs).  200-300 bp adaptor ligated nucleic acid was gel purified and enriched via 19 cycles of PCR amplification with Phusion High-Fidelity DNA Polymerase, followed by sequencing on an Illumina HiSeq 2000 system
GEO Accession
GSM1163119