GSM4677708: CD_H3K36me3_ChIP-Seq_R2; Mus musculus; ChIP-Seq
ESCs were harvested and MEF-depleted. Chromatin was prepared by cross-linking cells with 1% formaldehyde for 5 min, followed by adding glycine to 0.125M for 5 min. Cells were washed and resuspended in ChIP Lysis Buffer (50mM Tris-HCl pH8.0, 20mM EDTA,1% SDS, protease inhibitor cocktail) and sonicated using Covaris S220 for 14 cycles of alternating 45s on/off with peak power = 170, duty factor = 5, cycles per burst = 200, 10-13x106 cells at a time. Soluble chromatin was collected by centrifuging at 12,000 x g for 10 min. 20ug of chromatin was used for each sample, together with a 0.4ug spike-in of human chromatin from 293T cells (50:1 of sample to spike-in chromatin ratio). The combined chromatin was diluted with 9 parts volume of Dilution Buffer (16.7mM Tris pH8.0, 0.01% SDS, 1.1% Triton-X-100, 1.2mM EDTA, 167mM NaCl), and incubated with 3ug of H3K36me3 antibody (Active Motif, 61101) overnight at 4°C. Equal portions of protein A and protein G magnetic beads (Thermo Fisher Scientific, 1002D and 1004D, respectively) were washed and added to each sample for 2 hrs at 4°C. Following IP, beads were washed twice with low salt buffer (50 mM HEPES, 0.1% SDS, 1% Triton X-100, 0.1% deoxycholate, 1 mM EDTA, and 140 mM NaCl), high salt buffer (50 mM HEPES, 0.1% SDS, 1% Triton X-100, 0.1% deoxy- cholate, 1 mM EDTA, and 500 mM NaCl), LiCl buffer (20 mM Tris, 0.5% NP-40, 0.5% deoxycholate, 1 mM EDTA, and 250 mM LiCl), and TE. Elution of DNA was performed by incubating beads with elution buffer (50 mM Tris, 1 mM EDTA, and 0.1% SDS) followed by reverse cross-linking at 65°C overnight with 1% SDS. Samples were treated with RNase A for 30 min followed by 2 h of proteinase K digestion. Magnetic beads were removed, and DNA was purified by phenol:chloroform extraction followed by ethanol precipitation. Libraries were prepared using the Ovation Ultralow System V2 (NuGen, 0344) according to manufacturer's protocol
GEO Accession
GSM4677708