title: GSM2339531: H3K9me3 ChIP-Seq in MEFs Kap1 KO cells; Mus musculus; ChIP-Seq
library_construction_protocol: Cells were washed with PBS, fixed for 10 minutes at 1% formaldehyde, and quenched with glycine (at 125 mM final) for 5 minutes at room temperature. Cells were washed three times with ice-cold PBS, and harvested. The pellet was lysed, resuspended in 1 mL of sonication buffer (10 mM Tris at pH 8, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% NaDOC, 0.5% NLS, and protease inhibitors), transferred to TC 12x12 tubes (Covaris), and sonicated (Covaris settings: 30 minutes, 5% duty cycle, 140W, 200 cycles). Chromatin was decrosslinked (RNAse A at 1μg/μL, 65°C overnight), purified and quantified by Nanodrop. Fragment size was assessed on a Bioanalyzer High Sensitivity chip (Agilent 2100). Immunoprecipitations were performed with 40 ug of chromatin (for KAP1 and SETDB1), or 20 ug of chromatin  (for histone modifications), with antibody-coupled magnetic beads (Dynabeads, ThermoFisher) in IP buffer (10 mM Tris at pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 2% Triton X-100, and protease inhibitors) overnight. Chromatin was decrosslinked (Proteinase K at 400ng/μL, 65°C overnight) and DNA purified for analysis. Illuminal ChIP-seq protocol
attributes.tag: GEO Accession
attributes.value: GSM2339531