Sample information curated by ChIP-Atlas

Antigen

Antigen Class
TFs and others
Antigen
STAG1

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 Stem and Progenitor Cell
cell type
HSPC
chip antibody
SA1
antibody source
Abcam, ab4457

Sequenced DNA Library

library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
ChIP assays were performed as previously described with minor variations (Steiner, L. A., Maksimova, Y., Schulz, V., Wong, C., Raha, D., Mahajan, M. C., Weissman, S. M., and Gallagher, P. G. (2009) Mol Cell Biol). 1 × 107 cells were cross-linked with 1% formaldehyde for 10 min at room temperature, followed by Dounce homogenization. Cross-linked nuclei were isolated, followed by sonication with a Covaris S2 shearing device (duty cycle 10%, intensity 5, 15 minutes), to obtain chromatin-containing DNA fragments with an average size of ~200-400bp. For each ChIP, 20 μg of antibody was used. Samples were immunoprecipitated with antibodies against antibody against CTCF (Millipore,07-729), the SA-1 subunit of cohesin (Abcam, ab4457), trimethyl histone 3 lysine 27 (Abcam ab6002) or nonspecific rabbit IgG (sc-2091, Santa Cruz). The antigen-antibody complex was captured on protein G beads, washed four times with radioimmunoprecipitation assay buffer, and then washed with phosphate-buffered saline. The DNA-protein complex was eluted from the protein G beads with 1% sodium dodecyl sulfate at 65°C, and cross-linking of DNA-protein adducts was reversed by overnight incubation at 65°C. After proteinase K and RNase digestion of the reverse-cross-linked sample, DNA was extracted with phenol-chloroform, precipitated with ethanol, then cleaned with the QIAquick PCR Purification Kit (Qiagen) according to manufacturer instructions. ChIP libraries were prepared using the KAPA Library Preparation Kit (KAPA Biosystems) per the manufacturer's protocol. Briefly, ChIP DNA was end repaired, followed by addition of an adenine base at the 3' and NEXTflex ChIP-Seq Barcodes (BiooScientific) adaptor ligation. The modified DNA was then size selected on a 2% agarose and purified by using a gel extraction kit (Qiagen). One microliter of size-selected adaptor ligated DNA was initially analyzed by SYBR Green Q-PCR to determine the number of cycles required to reach 50% of complete amplification. Adaptor ligated DNA was PCR amplified with one initial heating step of 98°C for 45 second, followed by the pre-determined number of cycles with a melting temperature of 98°C for 15 s, an annealing temperature of 65°C for 30 s, and a product extension at 72°C for 30 s. At the end of the amplification, a final extension at 72°C for 1 min was performed. Amplified ChIP DNA was subjected to deep sequencing using standard Yale Center for Genome analysis protocols.

Sequencing Platform

instrument_model
Illumina HiSeq 2000

hg19

Number of total reads
33635225
Reads aligned (%)
89.9
Duplicates removed (%)
6.7
Number of peaks
17035 (qval < 1E-05)

hg38

Number of total reads
33635225
Reads aligned (%)
90.5
Duplicates removed (%)
6.2
Number of peaks
16885 (qval < 1E-05)

Base call quality data from DBCLS SRA