Sample information curated by ChIP-Atlas

Antigen

Antigen Class
Input control
Antigen
Input control

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
Mouse Embryonic Fibroblasts
tissue
Mouse Embryonic Fibroblasts
strain
129S1/SvImJ
genotype
Wild type
media
DMEM supplemented with L-glutamine, Na-pyruvate, β-mercaptoethanol, penicilin-streptomycin, 10% FBS
media supplement
NA
treatment days
NA
antibody
NA
fixation strategy
formaldehyde
sonication strategy
Covaris S220

Sequenced DNA Library

library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
5-10x106 cells were cross-linked with 1% paraformaldehyde for 10 min and stopped by adding Glycine (to be 125 mM). The cross-linked material was washed with buffer 1 (0.25% Triton, 10 mM EDTA, 0.5 mM EGTA, 10 mM Tris pH7.5) and Buffer 2 (200 mM NaCl, 10 mM EDTA, 0.5 mM EGTA, 10 mM Tris pH7.5) supplemented with protease inhibitor cocktail (Roche). Cell pellet was lysed in SDS lysis buffer (50 mM Tris pH 8.0, 10mM EDTA, 1% SDS, 50 mM PMSF) and was sonicated to generate 200 to 600 bp fragments. Fragmented chromatin was immunoprecipitated with magnetic beads coupled with 5 μg of antibody. The beads were washed with low salt buffer (50 mM Tris pH 8.0, 150 mM NaCl, 0.1% SDS, 0.5% Deoxycholate, 1% NP40, 1 mM EDTA), high salt buffer (50 mM Tris pH 8.0, 500 mM NaCl, 0.1% SDS, 0.5% Deoxycholate, 1% NP40, 1 mM EDTA), LiCl wash buffer (50 mM Tris pH 8.0, 250 mM LiCl, 0.5% Deoxycholate, 1% NP40, 1 mM EDTA), Morohashi RIPA buffer (50mM Tris pH7.5, 150mM NaCl, 5mM EDTA, 0.5% NP40, 0.1% SDS), DOC/Triton Buffer (25mM Tris pH7.5, 150mM NaCl, 5mM EDTA, 1% Triton-X-100, 0.5% DOC) and Tris-EDTA buffer (10 mM Tris pH 8.0, 1 mM EDTA) for 10 min at 4 °C. Complexes were eluted in elution buffer (1% SDS, 0.1 M NaHCO3) for 2 h at 65 °C and reverse cross-linked for 6 h at 65°C. Reverse cross-linked DNA was purified by QIAquick PCR purification kit ChIP DNA samples for sequencing were prepared as described for ChIP above. PCR cleanup with MinElute PCR Purification kits (Qiagen) yielded 20 μl ChIP DNA which was used immediately in a 23.3 μl combined end repair and A-tailing reaction using a KAPA Hyper Prep Kit (Kapa Biosciences) for 30 minutes at 20°C followed by 30 minutes at 65°C. 10 μl of ligase buffer, 3.7 μl of Y-Adapters diluted 1:50 (Illumina 5179206) and 3.3 μl ligase (KAPA Hyper Prep Kit) were added and incubated at 20°C for 4 hours. Double-stranded DNA fragments were purified from this reaction using KAPA Pure Beads (Kapa Biosciences) and eluted in 22 μl 10 mM Tris pH8.0. The amplification cycle number was determined empirically by using 1 μl of the eluent in a 10 μl test reaction containing a 1:10,000 dilution of SYBR Green I (Invitrogen), 2x KAPA HotStart ReadyMix (Kapa KM2618 and custom barcoding primers. An appropriate number of cycles was used in a subsequent 50 μl reaction with the remaining eluent in the absence of SYBR Green. Libraries were size-selected on a 2% E-Gel EX agarose gel (Invitrogen) and fragments between 150 and 350 bp were extracted using a QIAEX II Gel Extraction Kit (Qiagen) performed at room temperature. Libraries were quantitated with a high sensitivity Qubit dsDNA HS Assay Kit (Invitrogen Q32854) and pooled prior to submission quality control analyses and next-generation sequencing.

Sequencing Platform

instrument_model
Illumina HiSeq 2500

mm10

Number of total reads
16176317
Reads aligned (%)
96.3
Duplicates removed (%)
6.4
Number of peaks
232 (qval < 1E-05)

mm9

Number of total reads
16176317
Reads aligned (%)
96.2
Duplicates removed (%)
6.7
Number of peaks
169 (qval < 1E-05)

Base call quality data from DBCLS SRA