Sample information curated by ChIP-Atlas

Antigen

Antigen Class
TFs and others
Antigen
Epitope tags

Cell type

Cell type Class
Pluripotent stem cell
Cell type
ES cells
NA
NA

Attributes by original data submitter

Sample

source_name
mouse embryonic stem cells
cell type
E14
genotype
Tip60-H3F
chip antibody
Flag-M2 (Sigma, F1804)
protocol
RNaseH1 overexpression

Sequenced DNA Library

library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
Cells from ~80 % confluent 10 cm dishes were crosslinked by adding fixation solution (1% formaldehyde, 0.1M NaCl, 1mM EDTA, 50mM HEPES⋅KOH pH 7.6) for 10 minutes at room temperature. Crosslinking was quenched with 125mM Glycine for 5 minutes. Cells were washed twice with cold PBS containing protease inhibitors (Roche), and pelleted at 1000g for 5 minutes at 4°C. Cell pellets were either flash frozen in liquid nitrogen and stored at -80°C or immediately sonicated. Pellets were resuspended in Lysis buffer 1 (50mM HEPES⋅KOH pH 7.6, 140mM NaCl, 1mM EDTA, 10% (v/v) Glycerol, 0.5% NP-40, 0.25% Triton X-100) including protease inhibitors and incubated for 10 minutes at 4°C. After centrifugation at 1350g for 5 minutes, pellets were resuspended in Lysis buffer 2 (10mM Tris-HCl pH 8.0, 200mM NaCl, 1mM EDTA, 0.5mM EGTA) containing protease inhibitors and incubated for another 10 minutes at 4°C. Pellets were collected after centrifugation at 1350g for 5 minutes and resuspended in Lysis buffer 2 for sonication. Samples were transferred into 15 ml tubes (Falcon) and sonicated in a Bioruptor set to high for 2 cycles (10 minutes for one cycle with 30 sec on/ 30 sec off). The supernatants were collected after a 13000 rpm spin for 10 minutes at 4°C. 50μl Protein G Magnetic beads (NEB) were washed twice with PBS with 5mg/ml BSA and 10μg of anti-Flag M2 antibody (Sigma) coupled in 500μl PBS with 5mg/ml BSA overnight at 4°C. Immunoprecipitation was performed with antibody-coupled beads and sonicated supernatants in ChIP buffer (20mM Tris-HCl pH8.0, 150mM NaCl, 2mM EDTA, 1% Triton X-100) overnight at 4°C. Magnetic beads were washed twice with ChIP buffer, once with ChIP buffer including 500mM NaCl, 4 times with RIPA buffer (10mM Tris-HCl pH8.0, 0.25M LiCl, 1mM EDTA, 0.5% NP-40, 0.5% Na⋅Deoxycholate), and once with TE buffer (pH 8.0). Chromatin was eluted twice from washed beads by adding elution buffer (20mM Tris-HCl pH8.0, 100mM NaCl, 20mM EDTA, 1% SDS) and incubating for 15 minutes at 65°C. The crosslinking was reversed at 65°C for 6hr and RNase A (Sigma) was added for 1hr at 37°C followed by proteinase K (Ambion) treatment overnight at 50°C. ChIP-enriched DNA was purified using Phenol/Chloroform/Isoamyl alcohol extractions in phase-lock tubes. Libraries were prepared according to Illumina's instructions. Briefly, DNA was end-repaired using a combination of T4 DNA polymerase, E. coli DNA Pol I large fragment (Klenow polymerase) and T4 polynucleotide kinase. The blunt, phosphorylated ends were treated with Klenow fragment (exo minus) and dATP to yield a protruding 3- 'A' base for ligation of barcoded adapters which have a single 'T' base overhang at the 3’ end. DNA purification on Zymo Research PCR purification columns (Zymo, Irvine, CA) was performed following each enzyme reaction. The adaptor-ligated material was then PCR amplified with Phusion polymerase using 16 cycles of PCR before size selection of 200-300 bp fragments on a 2% agarose gel. Libraries with different barcodes were pooled together and single-end sequencing (50 bp) was performed on an Illumina HiSeq2000.

Sequencing Platform

instrument_model
Illumina HiSeq 2000

mm10

Number of total reads
26967983
Reads aligned (%)
96.1
Duplicates removed (%)
32.7
Number of peaks
522 (qval < 1E-05)

mm9

Number of total reads
26967983
Reads aligned (%)
95.9
Duplicates removed (%)
32.7
Number of peaks
541 (qval < 1E-05)

Base call quality data from DBCLS SRA