GSM2339531: H3K9me3 ChIP-Seq in MEFs Kap1 KO cells; Mus musculus; ChIP-Seq
Sample information curated by ChIP-Atlas
Antigen
Antigen Class
Histone
Antigen
H3K9me3
Cell type
Cell type Class
Embryonic fibroblast
Cell type
MEF
Tissue
Embryonic Fibroblast
Lineage
primaryCells
Description
Mouse Embryonic Fibroblast
Attributes by original data submitter
Sample
source_name
H3K9me3 ChIP-Seq in MEFs Kap1 KO cells
strain background
C57BL/6
genotype/variation
Kap1 KO
developmental stage/age
embryonic
tissue/cell type
Mouse embryonic fibroblast (MEF)
chip antibody
antiH3K9me3 (Diagenode, C15410056)
Sequenced DNA Library
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
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