Sample information curated by ChIP-Atlas

Antigen

Antigen Class
TFs and others
Antigen
Pcgf1

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 line
E14
chip antibody
Pcgf1
genotype
Ring1a-/-_Ring1bfl

Sequenced DNA Library

library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
ChIP assays were carried out as described previously (Ferrari et al., 2014) . Briefly, 1% formaldehyde cross- linked chromatin (1 mg) was fragmented by sonication to an average size of 300–600 bp and incubated overnight in IP Buffer (33 mM Tris-HCl, pH 8, 100 mM NaCl, 5 mM EDTA, 0.2% NaN 3 , 0.33% SDS, 1% Triton X-100) at 4ºC with 1–8 µg of the indicated antibodies, followed by incubation for 2–4 hr with protein A sepharose beads (GE Healthcare). Beads were washed three times with 150 washing buffer (20 mM Tris/HCl, pH 8, 150 mM NaCl, 2 mM EDTA, 0.1% SDS, 1 % Triton X-100), once with 500 wash buffer (20 mM Tris/HCl, pH 8, 500 mM NaCl, 2 mM EDTA, 0.1% SDS, 1% Triton X-100), and re-suspended in 120 µl of de-crosslinking solution (0.1 M NaHCO3, 1% SDS). For ChIP-seq, column-purified DNA from a ChIP experiment was quantified using the Qubit dsDNA HS Assay Kit (Thermo Scientific, Q32854) and 10 ng DNA were processed at IEO NGS core unit employing an automated platform (Beckman Coulter) with the Illumina ChIP-seq sample prep kit (IP-102-1001) and multiplexing oligonucleotide kit (PE400-1001). DNA libraries were quality-checked and quantified on an automated sample processing workstation (Caliper Life Sciences) and used for cluster generation and sequencing by the HiSeq 2000 (Illumina) and 50 bp read length. For PCGF1 ChIP, chromatin was crosslinked for 50 min at RT with 2 mM EGS (Sigma) and then for 10 min with 1% formaldehyde. For histone modifications, quantitative ChIP experiments were performed relative to a reference exogenous genome (ChIP-Rx) (Orlando et al., 2014) . For this, a total of 5% of Drosophila chromatin from S2 cell line, with a sheared size of 200–300 bp, was added to each ChIP reaction. RNA-seq was performed with minor modifications according to the SMART-seq2 protocol (Picelli et al., 2014) . Briefly, poly-A containing mRNA molecules from 2 g of total extracted RNA were copied into first- strand cDNA by reverse transcription and template-switching using oligo(dT) primers and an LNA- containing template-switching oligo (TSO); resulting cDNA was pre-amplified, purified, and tagmented with in-house produced Tn5 transposase. cDNA fragments generated after tagmentation were gap-repaired, amplified by PCR, and cleaned to obtain the final cDNA library.

Sequencing Platform

instrument_model
Illumina HiSeq 2000

mm10

Number of total reads
45706708
Reads aligned (%)
89.3
Duplicates removed (%)
8.0
Number of peaks
4733 (qval < 1E-05)

mm9

Number of total reads
45706708
Reads aligned (%)
89.1
Duplicates removed (%)
8.0
Number of peaks
4741 (qval < 1E-05)

Base call quality data from DBCLS SRA