Sample information curated by ChIP-Atlas

Antigen

Antigen Class
TFs and others
Antigen
EPAS1

Cell type

Cell type Class
Cardiovascular
Cell type
HUVEC
Primary Tissue
Umbilical Cord
Tissue Diagnosis
Normal

Attributes by original data submitter

Sample

source_name
HUVEC cells
cell line
HUVEC
chip antibody
EPAS1 (PM9)
passagges
from 4 to 6

Sequenced DNA Library

library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
HUVEC cells fixed and protein-DNA complexes crosslinked by treatment with 1% formaldehyde, and then quenched with glycine (125mM). Next, cells were washed 3 times with ice-cold PBS, collected using a cell scraper and resuspended in ice-cold lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl pH 8.1). DNA was sheared by using a sonicator (Diagenode Bioruptor, Belgium). Samples were diluted 1/10 with dilution Buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl pH 8.1, 167 mM NaCl) and then precleared with Salmon Sperm DNA/Protein A agarose 50% slurry (Fast Flow, Millipore, 16-156.) Sample were immunoprecipitated with different antibodies at 4°C rocking overnight; HIF1A (PM14), EPAS1 (PM9), [57] ARNT (Novus NB-100-110) and pre-immune serum used as negative control.Immunocomplexes were recovered by addition of Salmon Sperm DNA/Protein A agarose 50% slurry. Then samples were sequentially washed in Low Salt Wash Buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.1, 150 mM NaCl), High Salt Wash Buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.1, 500 mM NaCl), LiCl buffer (0.25 M LiCl, 1% NP-40, 1% NaDesoxycholate, 1 mM EDTA, 10 mM Tris-HCl pH 8.1), and twice in TE buffer (10 mM Tris-HCl pH 8, 1 mM EDTA). Elution of protein-bound DNA was performed using 500 µl of fresh elution buffer (1% SDS, 0.1 M NaHCO3). Next crosslinking of all samples was reversed by the overnight incubation with 200 mM NaCl at 65°C. Next day, proteins were removed by the addition of 10 µl of 0.5 M EDTA, 20 µl Tris-HCl pH 6.5 and proteinase K (40 µg/sample). Then immunoprecipitated DNA was purified by Phenol:Chloroform:Isoamyl alcohol extraction and ethanol precipitation (UltraPure™ phenol:Chloroform:Isoamyl Alcohol 25:24:1, Invitrogen, 15593-031). Libraries were prepared according to Illumina ChIP-Seq kit's intructions (Illumina, IP-202-1012).

Sequencing Platform

instrument_model
Illumina HiSeq 2000

hg19

Number of total reads
25590109
Reads aligned (%)
63.4
Duplicates removed (%)
56.4
Number of peaks
90 (qval < 1E-05)

hg38

Number of total reads
25590109
Reads aligned (%)
64.9
Duplicates removed (%)
55.8
Number of peaks
111 (qval < 1E-05)

Base call quality data from DBCLS SRA