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
cell type
MEF-derived cells
strain
C57BL/6
transduction
ZFATF1, ZFATF2, ZFATF3, Sox2, Klf4, c-Myc/Empty, Sox2, Klf4, c-Myc
chip antibody
none (input)

Sequenced DNA Library

library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
For histone marks, 6 x 10^6 cells were harvested, and for ATFs, 2.5 x 10^7 cells were harvested. Cells were fixed in 1.5% formaldehyde, then flash frozen. Cells were lysed with a series of 3 buffers: LB1, LB2, and LB3. LB1 consisted of 10 mM HEPES, 10 mM EDTA, 0.5 mM EGTA, and 0.25% Triton X-100. LB2 consisted of 200 mM NaCl, 10 mM HEPES, 1 mM EDTA, and 0.5 mM EGTA. LB3 consisted of 50 mM Tris-HCl, 10 mM EDTA, 0.5% Empigen BB, and 1% SDS. Samples were sonicated in a Misonix sonicator (S-4000) at 60% power, 10 sec on and 10 off, for a total of 32 min total pulse time. Samples were cleared by centrifugation 17,000 x g for 10 min. For preclearing, samples were incubated with magnetic Protein G beads (Life Technologies #10003D) for 1 hour at 4°C. After preclearing, 1% of sample was saved as input. Samples were incubated with the appropriate antibody, H3K27ac antibody (Abcam #ab4729), H3K9me3 antibody (Abcam #ab8898), or HA antibody (Abcam #ab9110), overnight at 4°C with IP Buffer (2 mM EDTA, 150 mM NaCl, 20 mM Tris-HCl, and 1% Triton X-100). Protein-DNA complexes on magnetic beads were washed in WB1 once, WB2, once, WB3 once, and TE Buffer twice. WB1 consisted of 2 mM EDTA, 20 mM Tris-HCl, 0.1% SDS, 1% Triton X-100, 150 mM NaCl). WB2 consisted of 2 mM EDTA, 20 mM Tris-HCl, 0.1% SDS, 1% Triton X-100, and 500 mM NaCl. WB3 consisted of 1 mM EDTA, 10 mM Tris-HCl, 250 mM LiCl, 1% deoxycholate, and 1% NP-40. DNA was eluted in 0.1 M NaHCO3, 0.2 M NaCl, and 1% SDS. Protein-DNA complexes were reverse crosslinked by incubation at 65°C for 6 hours. DNA was treated with RNase A and Proteinase K. Captured DNA was column purified (Epoch Life Sciences #1920-250). Samples were prepared for sequencing with the TruSeq ChIP Sample Preparation Kit (Illumina #IP-202-1012) as per the manufacturer instructions and quantified with a Qubit fluorometer (Life Technologies #Q32866). Three, four or six TruSeq indexed ChIP samples were pooled per lane. All samples were loaded at a final concentration of 8 pM and sequenced as single reads on the Illumina HiSeq 2500 following the manufacturer's protocols.

Sequencing Platform

instrument_model
Illumina HiSeq 2500

mm10

Number of total reads
76982478
Reads aligned (%)
98.3
Duplicates removed (%)
16.7
Number of peaks
609 (qval < 1E-05)

mm9

Number of total reads
76982478
Reads aligned (%)
98.0
Duplicates removed (%)
16.6
Number of peaks
683 (qval < 1E-05)

Base call quality data from DBCLS SRA