Sample information curated by ChIP-Atlas

Antigen

Antigen Class
Input control
Antigen
Input control

Cell type

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

Attributes by original data submitter

Sample

source_name
WT_nonTsh_ChIP_input
cell line
v6.5
cell type
embryonic stem cell
genotype/variation
Wildtype/WT
shRNA
nonTsh

Sequenced DNA Library

library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
RNA was extracted using Trizol reagent (Life technologies). RNAse free DNaseI (Sigma) was used to eliminate DNA contamination and the treated RNA was purified with RNeasy mini kit (Qiagen). For ChIP, ESCs were fixed with 1% formaldehyde for 10 minutes. After quenching and cell lysis, chromatin were sonicated using a E220 focused-ultrasonicator (Covaris). 100 µg sheared chromatin, 5 µg antibody, and 50 µl protein A/G beads (Santa Cruz) were used for each immunoprecipitation. Immunoprecipitated DNA were purified after washing, eluting, and reverse-croslinking and submitted for library preparation. 4C-seq was performed following the protocol published in (van de Werken et al. 2012). In brief, cross-linked chromatin was digested with DpnII, ligated under diluted conditions. Cross links were reversed and DNA was further digested by NlaIII and again ligated under diluted conditions.For each viewpoint approximately 3.2 μg of the 4C-seq library was amplified using 16 individual PCR reactions with inverse primers containing Illumina adaptor sequence. Multiplexed samples were sequenced on the Illumina Nextseq500 system using 75 bp single-end reads according to the manufacturer’s specifications. ChIP-seq libraries were prepared using the KAPA HTP Library Preparation Kit complemented with NEXTflex DNA Barcodes from Bioo Scientific. 10 ng of DNA was used as starting material for input and ip samples. Libraries were amplified using 13 cycles on the thermocycler. Post amplification libraries were size selected at 250- 450bp in length using Agencourt AMPure XP beads from Beckman Coulter. Libraries were validated using the Agilent High Sensitivity DNA Kit. RNA-seq libraries were prepared using the Illumina TruSeq Stranded Total RNA Preparation Kit with Ribo-Depletion. Input RNA quality was validated using the Agilent RNA 6000 Nano Kit. 200ng-1 µg of total RNA was used as starting material. Libraries were validated using the Agilent DNA 1000 Kit

Sequencing Platform

instrument_model
NextSeq 500

mm10

Number of total reads
46671482
Reads aligned (%)
96.4
Duplicates removed (%)
16.8
Number of peaks
593 (qval < 1E-05)

mm9

Number of total reads
46671482
Reads aligned (%)
96.1
Duplicates removed (%)
16.8
Number of peaks
664 (qval < 1E-05)

Base call quality data from DBCLS SRA