Sample information curated by ChIP-Atlas

Antigen

Antigen Class
TFs and others
Antigen
PAX7

Cell type

Cell type Class
Pluripotent stem cell
Cell type
hESC H9
NA
NA

Attributes by original data submitter

Sample

source_name
GFP+ sorted cells collected at day 23
cell type
H9 cells
doxycycline-treatment
dox-treated from (day 10 to 23)
collection time
day 23 of differentiation
chip antibody
PAX7 (Developmental Studies Hybridoma Bank)

Sequenced DNA Library

library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
ChIP-seq: Cell pellets were incubated in lysis buffer LB1 supplemented with protease inhibitors (50mM HEPES KOH pH7.5, 140mM NaCl, 1mM EDTA, 10% glycerol, 0.5% NP40, 0.25% Triton X-100 + Complete-mini - Roche) for 10 minutes at +4°C followed by incubation in buffer LB2 supplemented with protease inhibitors (10mM TRIS HCl pH 8, 200mM NaCl, 1mM EDTA, 0.5mM EGTA + Complete-mini - Roche) for 10 minutes at +4°C. Cell pellet was then resuspended in LB3 supplemented with protease inhibitors (10mM TRIS HCl pH 8, 100mM NaCl, 1mM EDTA, 0.5mM EGTA, 0.1% Sodium Deoxycholate, 0.5% N-lauroylsarcosine + Complete-mini - Roche) and then sonicated with a Branson sonicator at 18% power for 1 minute with intervals of 10 sec ON-10 sec OFF. Each sample was subjected to 5-6 cycles of sonication to reach an average chromatin size of 300bp. After shearing, samples were centrifuged for 10 minutes at 16000g and snap frozen in liquid nitrogen if not processed immediately. For each ChIP, 20µg of chromatin (diluted to 400µl) were precleared for 4h at 4°C with 15µl of BSA-blocked Protein G-conjugated sepharose beads (GE healthcare) and then supplemented with 1/10 volume of 10% Triton X-100. Immunoprecipitation was performed by overnight incubation with normal mouse IgG (8µg – Santa Cruz Biotechnology) or anti-Pax7 antibody (1:50 – Developmental Studies Hybridoma Bank). Immune complexes were recovered by incubation with 15µl of BSA-blocked Protein G-conjugated sepharose beads for 4h at 4°C and then washed 5 times with RIPA wash buffer (50mM HEPES KOH pH7.5, 500mM LiCl, 1mM EDTA, 1% NP40, 0.25% Triton X-100, 0.7% Sodium Deoxycholate) and one time with TEN buffer (10mM TRIS HCl pH 8, 1mM EDTA, 50mM NaCl). Immunoprecipitated chromatin was recovered by incubating beads with 200µl of Elution buffer (50mM TRIS HCl pH 8, 10mM EDTA, 1% Sodium Dodecyl Sulfate) for 20 minutes at 65°C. Chromatin from IP and Input (equivalent to 1% of starting material) was reverse crosslinked overnight at 65°C, then diluted 1:1 with TE (10mM TRIS HCl pH 8, 1mM EDTA) supplemented with 4µl of RNaseA 20mg/ml and incubated for 2 hours at 37°C followed by Proteinase K treatment (4µl of 20mg/ml stock for each sample) for 30 minutes at 55°C. DNA was purified by Phenol-chloroform-isoamyl alcol extraction (twice) followed by chloroform extraction, then supplemented with 1/10 of volume of 3M Sodium Acetate pH 5 and 1.5µl of Glycogen and precipitated with 2 volumes of 100% Ethanol at -80°C for >1 hour. Followed 30 minutes centrifuge at 16000g, pellet were washed with 75% ethanol, air dried and dissolved in 45µl H2O. ChIP-seq: Libraries were generated following a gel-free protocol using AMPure XP beads (Beckman Coulter) for all the purification and size selection steps. 10ng or less of DNA were end repaired using End-it DNA end repair (Epicentre), then A-tailed using Klenow Fragment (3'→5' exo- NEB) followed by adapter-barcode ligation using T4 DNA ligase (Enzymatics). Illumina compatible adapter-barcodes were purchased from BIOO scientific. After ligation, DNAs were negatively size selected using 0.5x Ampure XP beads and unbound DNAs were positively size selected by adding 0.4x Ampure XP beads (this step allows for retention of DNA fragments ranging 200-500bp). Libraries were amplified using Phusion High Fidelity PCR master mix 2x (NEB) with a 16 cycles program.

Sequencing Platform

instrument_model
Illumina HiSeq 2500

hg19

Number of total reads
34472006
Reads aligned (%)
93.6
Duplicates removed (%)
3.2
Number of peaks
9506 (qval < 1E-05)

hg38

Number of total reads
34472006
Reads aligned (%)
95.2
Duplicates removed (%)
2.2
Number of peaks
9704 (qval < 1E-05)

Base call quality data from DBCLS SRA