GSM3406736: KO MEF 7 days H3K4me3 N5; Mus musculus; ChIP-Seq
Sample information curated by ChIP-Atlas
Antigen
Antigen Class
Histone
Antigen
H3K4me3
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
MEFs undergoing transdifferentiation
chip target
H3K4me3
chip antibody
Active motif 39159 (rabbit)
genotype
Kmt2b-/-
Stage
MEF 7 days
Sequenced DNA Library
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
Cells were fixed with PBS 1% formaldehyde. Fixation was quenched with 0,125M glycine. Cells were collected in SDS Buffer (100mM NaCl, 50mM Tris-HCl pH 8.1, 5mM EDTA pH 8, 0.5% SDS), centrifuged, then resuspended in 3 ml of ice-cold IP buffer (2 volumes SDS Buffer: 1 volume Triton Dilution Buffer) (Triton Dilution Buffer: 100mM NaCl, 100mM Tris-HCl pH 8.6, 5mM EDTA pH 8, 5% Triton X-100) and sonicated through the Digital Sonifier 450 (Branson) (ChIP-seq for Menin: 700-bp DNA fragments; ChIP-seq for H3K4me3 and H3K27me3: 200-bp DNA fragments). The immunoprecipitated product was purified through Protein G dynabeads (ThermoFisher Scientific, 10003D), afterwards washed with low-salt (150mM NaCl, 20mM Tris-HCl pH 8, 2mM EDTA pH 8, SDS 0.1%, 1% Triton X-100) and high-salt wash (500mM NaCl, 20mM Tris-HCl pH 8, 2mM EDTA pH 8, SDS 0.1%, 1% Triton X-100) buffer 3 times with 1 ml of 150 mM Wash Buffer and once with 1 ml of 500 mM Wash Buffer with the use of a Dynamag magnet (ThermoFisher Scientific, catalog number 12321D). Decrossilinking occurred through the incubation of beads (and the 1% input) with Decrosslinking Buffer (1% SDS, 0.1M NaHCO3) at 65 °C overnight. DNA was purified with the QiaQuick PCR Purification kit (Qiagen) following manufacturer's instructions. Libraries were prepared as previously described in Adamo et al., Nature Genetics 2015.