Sample information curated by ChIP-Atlas

Antigen

Antigen Class
Histone
Antigen
H3

Cell type

Cell type Class
Blood
Cell type
Hematopoietic Stem Cells
MeSH Description
Progenitor cells from which all blood cells derived. They are found primarily in the bone marrow and also in small numbers in the peripheral blood.

Attributes by original data submitter

Sample

source_name
human HSPCs
cell type
HSPCs
chip antibody
H3 : #ab1791
development stage
model leukemia cells from xenograft

Sequenced DNA Library

library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
[ChIP-seq] After crosslinking cells by using formaldehyde 1% (final concentration) for 7 minutes at room temperature, glycine 0.125nM (final concentration) was added for 5 minutes at room temperature to stop the reaction. Fixed cells were washed with PBS, resuspended in 1mL of a first lysis buffer (Triton 0.25%, Tris pH 8.0 10mM, EDTA 10mM, EGTA 0.5mM, protease inhibitor 1X) for 5 minutes, and in 1 mL of a second buffer for 30 minutes (NaCl 200mM, Tris pH 8.0 10mM, EDTA 1mM, EGTA 0.5mM, protease inhibitor 1X). Lysate was resuspended in another buffer (0.5% SDS, 0.5% Triton, Tris pH 8.0 10mM, NaCl 140mM, EDTA 1mM, EGTA 0.5mM, protease inhibitor 1X) and then sonicated by using a Covaris S2 sonicator. The parameters used were as follows: duty Factor 10%, 200 cycles per burst, 105W, 4 minutes. The supernatant collected after the spinning at 13000 rpm for 5 minutes was used for future experiments. Beads (Protein G Dynabeads™ ; resuspension buffer : Triton 1%, Tris pH 8.0 10mM, NaCl 150mM, EDTA 2mM) were incubated overnight on a rotator at 4°C with 3μg of antibodies recognizing histone marks (H3 : #ab1791, H3K4me3 : #ab8580-100, H3K27me3 : Millipore 07-449, H3K79me2 : #ab3594, H3K27ac : Millipore 07-360). Sonicated chromatin was incubated with 250μL of beads during 4 hours on a rotator at 4°C. The beads were washed sequentially with different buffers: 'Low Salt' Buffer (0.5% NP40, 15mM KCl, 10mM Tris pH 8.0, 1mM EDTA), 3 different 'High Salt' buffers (0.5% Triton, 10mM Tris pH 8.0, 100mM (2) or 400mM (3) or 500mM (4) NaCl), twice in LiCl Buffer (0.5% NP40, 250mM LiCl, 10mM Tris pH 8.0, 1mM EDTA). ChIPed material was then eluted a classic elution buffer (10mM Tris pH8.0, 1mM EDTA) and incubated overnight at 65°C with shaking (1200rpm). An RNase and proteinase K digestion was performed in order to reverse crosslink. DNA was extracted and purified by a classic phenol-chloroform protocol. [ATAC-seq] ATAC-seq experiment was based on method published by Buenrostro et al. (PMID: 24097267) TruSeq ChIP Library Preparation Kit by Illumina (#IP-202-9001) was used to construct DNA libraries as described in the manufacturer's instructions with a PCR for a total of 12 cycles using the Illumina indexed library primers. NextSeq 500 was used to sequence samples (75pb paired-end) according to standard Illumina protocols.

Sequencing Platform

instrument_model
NextSeq 500

hg38

Number of total reads
19137042
Reads aligned (%)
90.5
Duplicates removed (%)
15.2
Number of peaks
584 (qval < 1E-05)

hg19

Number of total reads
19137042
Reads aligned (%)
89.4
Duplicates removed (%)
15.6
Number of peaks
400 (qval < 1E-05)

Base call quality data from DBCLS SRA