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
Mouse embryonic fibroblasts
transgenes
flagMyod1 + rtTA
days post transgene induction
2 days
chip antibody
input

Sequenced DNA Library

library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
FLAG-tagged TFs were immunoprecipitated using FLAG M2 antibody conjugated beads (Sigma F2426). Approximately 60-100 million cells were used per replicate. Beads (~100uL slurry per sample) were washed thrice with IP dilution buffer (1% Triton X-100, 2mM EDTA pH8.0, 20mM Tris-HCl pH8.0, 150mM NaCl, 1mM DTT, 100uM PMSF), blocked overnight in IP dilution buffer with 0.1% BSA and 0.06% sheared salmon sperm DNA (Thermo Fisher; AM9680), then washed again three times with IP dilution buffer before use. Nuclei were isolated by incubating with cell lysis buffer (5mM HEPES pH7.9, 85mM KCl, 0.5% NP40, 100uM PMSF, protease inhibitors from Roche) for 10min on ice and centrifuged at 5000rpm for 5min at 4°C, then lysed with nuclear lysis buffer (50mM Tris-HCl pH8.0, 10mM EDTA pH8.0, 1% SDS, 100uM PMSF, protease inhibitors) for 10min on ice. Chromatin was sheared using either the Bioruptor (Diagenode) or Covaris sonicator until DNA was fragmented to 200-500bp. Sheared chromatin was diluted using 3X volume of IP dilution buffer and pre-cleared for at least 4hr at 4°C using IgG beads (Sigma A0919). 1% of pre-cleared chromatin was kept as input, and the remainder was incubated with FLAG beads overnight at 4°C. Beads were washed 8 times with IP wash buffer (20mM Tris-HCl pH8.0, 2mM EDTA pH8.0, 250mM NaCl, 1% NP40, 0.05% SDS, 100uM PMSF) and once with TE buffer with 100uM PMSF. Beads and 1% input samples were reverse cross-linked overnight in IP elution buffer (50mM NaHCO3, 1% SDS) at 65°C. Isolated DNA was RNase treated for 30min at 37°C, purified using QIAquick PCR purification columns (Qiagen) and eluted in DEPC water. ChIP-seq libraries were prepared using the NEBNext library prep kit (NEB; E6240) following the supplier's protocols with slight modifications. Adaptor ligation was performed using regular T4 ligase and ligase buffer. Size selection (200-500bp) was performing after adaptor ligation and PCR enrichment using 2% low-melt agarose gel (Lonza; 50080), and DNA was purified using the QiaQuick gel extraction columns from Qiagen.

Sequencing Platform

instrument_model
NextSeq 500

mm10

Number of total reads
18803871
Reads aligned (%)
97.7
Duplicates removed (%)
8.4
Number of peaks
384 (qval < 1E-05)

mm9

Number of total reads
18803871
Reads aligned (%)
97.5
Duplicates removed (%)
8.5
Number of peaks
397 (qval < 1E-05)

Base call quality data from DBCLS SRA