Sample information curated by ChIP-Atlas

Antigen

Antigen Class
TFs and others
Antigen
FOXA2

Cell type

Cell type Class
Pluripotent stem cell
Cell type
hESC derived mesendodermal cells
NA
NA

Attributes by original data submitter

Sample

source_name
in vitro derived endoderm
tissue
HUES64 derived day 5 endoderm
chip antibody
anti-FOXA2 (R&D, AF2400, ULB 0311101)

Sequenced DNA Library

library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
Cells collected by FACS were crosslinked in 1% formaldehyde for 15 minutes at room temperature, with constant agitation, followed by quenching with 125mM Glycine for 5 minutes at room temperature with constant agitation. Nuclei were isolated and chromatin was sheared using Branson sonifier until the majority of DNA was in the range of 200-700 base pairs. Chromatin was incubated with antibody overnight at 4°C, with constant agitation. Co-immunoprecipitation of antibody-protein complexes was completed using Protein A or Protein G Dynabeads for 1 hour 4°C, with constant agitation. ChIPs were completed using previously reported methods (Mikkelsen et al., 2010) ChIP-Bisulfite Sequencing (adapted from Brinkman et al, 2012) DNA was first subjected to end-repair in a 30-μl reaction containing 6 units T4 DNA polymerase, 2.5 units DNA Polymerase I (Large Klenow Fragment), 20 units T4 Polynucleotide Kinase (all New England Biolabs), dATP, dCTP, dGTP, and dTTP (0.125 mM each), and 1× T4 Ligase buffer with ATP for 30 min at 20°C. DNA was then adenylated in a 20-μl reaction containing 10 units Klenow Fragment (3′→5′ exo-) (New England Biolabs), 0.5 mM dATP and 1× NEB buffer 2 for 30 min at 37°C. DNA was then ligated to preannealed Illumina genomic DNA adapters containing 5-methylcytosine instead of cytosine (ATDBio) using T4 DNA ligase (New England Biolabs). Adapter-ligated DNA fragments were subsequently purified by phenol extraction and ethanol precipitation and size-selected on gel. 50 ng sheared and dephosphorylated Escherichia coli K12 genomic DNA was added to adapter-ligated DNA as carrier during size-selection and bisulfite conversion. DNA was run on 2.5% Nusieve 3:1 Agarose (Lonza) gels. Lanes containing marker (50 bp ladder; New England Biolabs) were stained with SYBR Green (Invitrogen), and size regions to be excised were marked with toothpicks and adapter-ligated DNA fragments from 200–400 and 400–550 bp were excised. DNA was isolated from gel using the MinElute Gel Extraction kit (QIAGEN). The low and high libraries were kept separate in subsequent steps. Adapter-ligated and size-selected DNA was subjected to two subsequent 5-h bisulfite treatments using the EpiTect Bisulfite kit (QIAGEN) following the manufacturer's protocol for DNA isolated from FFPE tissue samples. PCR amplification was done with 1.25 units Pfu Turbo Cx Hotstart DNA Polymerase (Stratagene), primer LPX 1.1 and 2.1 (0.3 μM each), dNTPs (0.25 mM each), 1× Turbo Cx buffer. Amplified libraries were purified with the MinElute PCR Purification kit (QIAGEN) and subsequently purified from gel essentially as described above; whole gels were stained with SYBR Green, and no carrier DNA was added. Final libraries were analyzed on analytical 4%–20% TBE Criterion precast gels (BioRad), and measured by Quant-iT dsDNA HS Assays (Invitrogen). CHIP:Cells collected by FACS were crosslinked in 1% formaldehyde for 15 minutes at room temperature, with constant agitation, followed by quenching with 125mM Glycine for 5 minutes at room temperature with constant agitation. Nuclei were isolated and chromatin was sheared using Branson sonifier until the majority of DNA was in the range of 200-700 base pairs. Chromatin was incubated with antibody overnight at 4°C, with constant agitation. Co-immunoprecipitation of antibody-protein complexes was completed using Protein A or Protein G Dynabeads for 1 hour 4°C, with constant agitation. ChIPs were completed using previously reported methods (Mikkelsen et al., 2010). Sequencing library production details can be found in the Supplemental Experimental Procedures. Sequencing libraries were submitted for sequencing on the Illumina Hiseq 2000. Immunoprecipitated DNA was end repaired using the End-It DNA End-Repair Kit (Epicentre), extended using a Klenow fragment (3’-5’ exo)(NEB), and ligated to sequencing adapter oligos (Illumina). Each library was then PCR-amplified using PFU Ultra II Hotstart Master Mix (Agilent), and a size range of 300-600 was selected for sequencing. RNA-Seq: Strand specific libraries were constructed as described in the main text using a strand specific method (Levin et al., 2010). Polyadenylated RNA was isolated using Oligo dT beads (Invitrogen) and fragmented to 200-600 base pairs, and then ligated to RNA adaptors using T4 RNA Ligase, (NEB), preserving strand of origin information. A primer that anneals to the RNA adaptor was used to facilitate cDNA synthesis, which was then followed by RNA degradation. The cDNA library was then ligated to a DNA adaptor, which was used for PCR enrichment of the library, with index sequences included in the primers used for amplification.

Sequencing Platform

instrument_model
Illumina HiSeq 2000

hg19

Number of total reads
49133422
Reads aligned (%)
73.7
Duplicates removed (%)
58.6
Number of peaks
31426 (qval < 1E-05)

hg38

Number of total reads
49133422
Reads aligned (%)
75.1
Duplicates removed (%)
57.9
Number of peaks
31595 (qval < 1E-05)

Base call quality data from DBCLS SRA