Sample information curated by ChIP-Atlas

Antigen

Antigen Class
TFs and others
Antigen
Myc

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
embryonic fibroblasts, E13.5
cell type
3T9 fibroblasts
genotype
c-mycflox/flox; in vitro infected with MycER
chip antibody
Myc N262 (Santa Cruz, sc-764)

Sequenced DNA Library

library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
Cells were washed in PBS and fixed for 15 min in 1% of formaldehyde (Sigma) in PBS. Glycin was added (125 mM glycin) and cells were incubated for 5 min. Cells were washed with PBS and scraped in SDS buffer (100mM NaCl, 50mM Tris pH 8.1, 5mM EDTA, 0.2% NaN3, 0.5% (w/v) SDS). Cells were cleared by centrifugation and the pellet resuspended in cold ChIP buffer (100mM NaCl, 133mM Tris pH 8.0, 5 mM EDTA, 0.2% NaN3, 0.67% SDS, 1.67% Triton X-100) supplemented with protease inhibitors (cOmplete Protease Inhibitor Cocktail Tablets, Roche) and phosphatase inhibitors (50 mM NaF, 1 mM β-GP, 1 mM NaV). All following steps were performed on ice, if not specified differently. Chromatin was sonicated to an average length of 200-400bp, pre-cleared with proteinG-sepharose beads (blocked with 0.5% E. coli tRNA (Sigma) and 0.5% BSA). 10µg of the following antibodies were used per ChIPseq reaction: Myc N262 (Santa Cruz, sc-764), RNAPII N20 (Santa Cruz, sc-899). The antibody was incubated overnight with 500-1000µl of chromatin. Blocked proteinG-sepharose beads were added, incubated for 2-3h and washed as follows: 3x mixed micelle buffer (150 mM NaCl, 20 mM Tris pH 8.1, 5 mM EDTA, pH 8.0, 5.2% sucrose, 0.02% NaN3, 1% Triton X-100, 0.2% SDS), 2x buffer 500 (50 mM HEPES pH 7.5, 0.1% sodium deoxycholate, 1 mM EDTA, 500 mM NaCl, 1% Triton X-100, 0.2% NaN3), 2x LiCl/detergent buffer (10 mM Tris pH 8.0, 0.5% sodium deoxycholate, 1 mM EDTA, 250 mM LiCl, 0.5% NP-40, 0.2% NaN3) and once in TE. DNA:protein crosslinking was reverted overnight (2% SDS in TE, 65°C), DNA purified using QiaQuick columns (Qiagen) and quantified using PicoGreen (Invitrogen). 2-5 ng of ChIP DNA were used for ChIPseq library preparation as described in (Blecher-Gonen et al., Nat Protoc, 2013) and sequenced with the Illumina HiSeq sequencer according to the manufacturer.

Sequencing Platform

instrument_model
Illumina HiSeq 2000

mm10

Number of total reads
50262465
Reads aligned (%)
94.6
Duplicates removed (%)
22.8
Number of peaks
23942 (qval < 1E-05)

mm9

Number of total reads
50262465
Reads aligned (%)
94.4
Duplicates removed (%)
22.7
Number of peaks
23974 (qval < 1E-05)

Base call quality data from DBCLS SRA