GSM5138724: H3K4me3_Meso48h_WT; Mus musculus; ChIP-Seq
Approximately 2*10^7 cells were cross-linked by addition of formaldehyde to 1% for 10 min at RT, quenched with 0.125 M glycine for 5 min at RT, and then washed twice in cold PBS. The cells were resuspended in Lysis Buffer 1 (50 mM Hepes-KOH pH 7.5, 140mM NaCl, 1mM EDTA, 10% Glycerol, 0.5% NP-40, 0.25% Triton X-100 and protease inhibitor) to disrupt the cell membrane and in Lysis Buffer 2 (10 mM Tris-HCl pH8.0, 200 mM NaCl, 1mM EDTA, 0.5 mM EGTA and protease inhibitor) to isolate nuclei. The isolated nuclei were then resuspended in SDS ChIP Buffer (20 mM Tris-HCl pH 8.0, 10 mM EDTA, 1% SDS and protease inhibitors). Extracts were sonicated using the BioruptorH Twin (Diagenode) for 2 runs of 10 cycles [30 sec ''ON'', 30 sec ''OFF''] at high power setting. Cell lysate was centrifuged at 12,000 g for 10 min at 4°C. The supernatant was diluted with ChIP Dilution Buffer (20 mM Tris-HCl pH 8.0, 150 mM NaCl, 2 mM EDTA, 1% Triton) before immunoprecipitation step. Streptavidin beads (Dynabeads®Protein G, Life Technologies) were saturated with PBS/1% BSA and the samples were incubated with 2 ug of antibody overnight at 4°C on a rotator.  Next day samples were incubated with saturated beads for two hours at 4°C on a rotator. Successively immunoprecipitated complexes were washed five times with RIPA buffer (50 mM Hepes-KOH pH7.6, 500mM LiCl, 1mM EDTA, 1% NP-40, 0,7% Na-Deoxycholate) at 4°C for 5 minutes each on a rotator. Elution Buffer was added and incubated at 65°C for 15 minutes. The decrosslinking was performed at 65°C overnight. De-crosslinked DNA was purified using QIAQuick PCR Purification Kit (QIAGEN) according to the manufacturer's instruction. Starting from 10 ng of ChIP eluted sample, the library was produced for the genome wide analysis following the NEBNext® ChIP-Seq Library Prep Reagent Set for Illumina® (E 6240L NEB) manufacturer's instructions. Libraries were sequenced on Illumina NextSeq 500 System (single-end 75 bp reads).