Sample information curated by ChIP-Atlas

Antigen

Antigen Class
Unclassified
Antigen
Unclassified

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
Mouse embryonic fibroblasts
genotype
WT
strain
C57BL/6

Sequenced DNA Library

library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
Cells were fixed with 1% formaldehyde (Sigma) for 10’ at 37°C. Fixation was quenched by addition of glycine (Sigma) at a final concentration of 125 mM. Twenty million fixed cells were washed with PBS and resuspended in 2 ml of RIPA buffer (10 mM Tris [pH 7.6], 1 mM EDTA, 0.1% SDS, 0.1% sodium deoxycholate, 1% Triton X-100, 1× Complete Mini EDTA free proteinase inhibitor (Roche)) or stored at −80°C until further processing. Sonication was performed using the Covaris S2 sonicator at duty cycle 20%, intensity 5, cycle/burst 200 for 30 min or using the Branson sonifier at amplitude 35%, 12 cycles of 20” sonication and 30” of pause. Ten microgram of respective antibody was incubated with 40 μl of Dynabeads Protein A (or G) for 15 min at room temperature. Antibody-bound beads were added to 1 ml of sonicated chromatin, incubated at 4°C overnight, and washed twice with RIPA buffer, twice with RIPA buffer containing 0.3M NaCl, twice with LiCl buffer (0.25 M LiCl, 0.5% Igepal-630, 0.5% sodium deoxycholate), once with TE (pH 8.0) plus 0.2% Triton X-100, and once with TE (pH 8.0). Crosslinking was reversed by incubating the beads at 65°C for 4 hr in the presence of 0.3% SDS and 1 mg/ml Proteinase K. ChIP DNA was purified using ChIP DNA Clean & Concentrator kit (Zymo). The library was prepared using the Ovation SP Ultralow library system (Nugen). 75 cycles of sequencing data were acquired on and Nextseq550 (Illumina). Antibodies for ChiP-seq were: anti-RAD21 (ab992, Abcam), anti-CTCF (07-729, Millipore), Topo IIβ Antibody (H-286) (sc13059, SantaCruz) and Topoisomerase II alpha antibody (EP1102Y) (ab52934, Abcam) libraries were prepared and sequenced following standard Illumina protocols

Sequencing Platform

instrument_model
NextSeq 550

mm10

Number of total reads
61936850
Reads aligned (%)
96.9
Duplicates removed (%)
44.3
Number of peaks
5549 (qval < 1E-05)

mm9

Number of total reads
61936850
Reads aligned (%)
96.6
Duplicates removed (%)
44.2
Number of peaks
5132 (qval < 1E-05)

Base call quality data from DBCLS SRA