Sample information curated by ChIP-Atlas

Antigen

Antigen Class
Input control
Antigen
Input control

Cell type

Cell type Class
Pluripotent stem cell
Cell type
hES BG01V
Primary Tissue
Embryo
Tissue Diagnosis
Normal

Attributes by original data submitter

Sample

source_name
embryonic cell line
cell line
BG01V - ATCC SCRC-2002
modifications or treatment
Transduced with a vector with biotin ligase sequence and a vector with the sequence of selected ZNF589 tagged with the biotinylation peptide
sample type
Input

Sequenced DNA Library

library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
DNA extraction: After selection, hESCs (10cm plate, ~80% confluence) were cross-linked in 1% formaldehyde for 10min, followed with 5min incubation with 125mM glycine. Next, the cells were washed 3x with ice cold PBS and scraped in PBS/0.2mM PMSF. The cell pellet was lysed in 500μl SDS ChIP Buffer (0.1% SDS, 1% Triton X-100, 2mM EDTA, 20mM Tris-HCl pH 8.1, 150mM NaCl, freshly supplemented with protease inhibitor cocktail from Sigma). After 10min incubation on ice, the chromatin was sonicated with 60 cycles (30s on + 30s off) in Bioruptor (Diagenode). The sonicated chromatin was centrifuged (10min, 4˚C, 12000rpm) and pre-purified on 50μl pre-washed Protein A Dynabeads (Life Technologies) (1h, 4˚C, 40rpm). Pre-purified chromatin was transferred on 50μl BSA-blocked (200μg/ul BSA in ChIP SDS Buffer for 3h followed by one wash in ChIP SDS Buffer) Streptavidin Dynabeads (Life Technologies) and incubated overnight (4˚C, 40rpm). Next, the beads were collected on a magnet and washed 2x with Buffer A (2% SDS), 1x Buffer B (0.1% deoxycholate sodium, 1% Triton X-100, 1mM EDTA, 50mM Hepes pH 7.5, 500mM NaCl), followed by 1x Buffer C (250mM LiCl, 0.5% NP40, 0.5% deoxycholate sodium, 1mM EDTA, 10mM Tris-HCl pH 8.1) and 2x TE (10mM Tris-HCl pH 7.5, 1mM EDTA). All washing steps were carried out in room temperature (8min, 40rpm) and in the presence of protease inhibitor cocktail (apart from the last washing in TE). After washing, the beads were resuspended in ChIP Elution Buffer (1% SDS, 10mM EDTA, 50mM Tris-HCl pH 8.1) and the chromatin was de-cross-linked (overnight, 65˚C). Next day, the eluted chromatin was separated from the beads and subjected to digestion with 5ng/µl RNase (30min at 37˚C) and 0.2µg/µl Proteinase K (2h at 55˚C) followed by column purification using ChIP DNA Clean and Concetrator kit (Zymo Research). DNA quantity was assessed with the picogreen dye using Qubit dsDNA HS Assay kit (Life Technologies) according to manufacturer’s protocol. RNA for RNA-seq analysis was isolated using TRIzol reagent solution according to manufacturer’s protocol. The RNA quantity and integrity number (RIN) was assessed using an Agilent BioAnalyzer 2000 (Agilent Technologies). The libraries for ChIP-seq were prepared with relevant library construction kits (NEB, Illumina, respectively) according to the manufacturer’s protocols.

Sequencing Platform

instrument_model
Illumina HiSeq 2500

hg38

Number of total reads
34056639
Reads aligned (%)
72.5
Duplicates removed (%)
9.7
Number of peaks
493 (qval < 1E-05)

hg19

Number of total reads
34056639
Reads aligned (%)
72.2
Duplicates removed (%)
10.4
Number of peaks
803 (qval < 1E-05)

Base call quality data from DBCLS SRA