Illumina HiSeq 2500 sequencing; ChIP-seq of ERG-bound regulatory elements in human and bovine vascular endothelial cells
Sample information curated by ChIP-Atlas
Antigen
Antigen Class
No description
Antigen
NA
Cell type
Cell type Class
Cardiovascular
Cell type
HUVEC
Primary Tissue
Umbilical Cord
Tissue Diagnosis
Normal
Attributes by original data submitter
Sample
ENA first public
2017-06-15
ENA last update
2016-10-17
ENA-CHECKLIST
ERC000011
External Id
SAMEA4515520
INSDC center alias
Scientist, Genetics & Genome Biology Program, SickKids Research Institute Assistant Professor, Department of Molecular Genetics, University of Toronto
INSDC center name
Scientist, Genetics & Genome Biology Program, SickKids Research Institute Assistant Professor, Department of Molecular Genetics, University of Toronto
INSDC first public
2017-06-15T17:02:09Z
INSDC last update
2016-10-17T14:21:08Z
INSDC status
public
Submitter Id
E-MTAB-5148:WL375
antibody part number
sc-353
broker name
ArrayExpress
cell type
HUVEC cell
common name
human
individual
12495
sample name
E-MTAB-5148:WL375
Sequenced DNA Library
library_name
WL375_s
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
Primary HUVECs were purchased from ScienCell. Primary BAECs were purchased from Cell Applications. HUVECs and HAECs were grown in supplier-recommended Endothelial Cell Growth Media (ScienCell) and cultured at 37° C in a 5%-CO2 humidified incubator. Approximately 20 million cells were used for the ERG and ~3 million cells for the H3K27ac ChIPs. All cells collected were between passage 3 and 6. Chromatin immunoprecipitation experiments were conducted as previously described in (Schmidt et al. 2009). Antibodies used for ChIP were mouse anti-H3K27ac (Millipore, 05-1334 monoclonal) and rabbit anti-ERG 1/2/3 (Santa Cruz Biotechnology, sc353 polyclonal). ChIP DNA was prepared for Illumina sequencing by blunt-end repair, dA-tailing, and ligation of Illumina adaptors using a NEBNext DNA library preparation kit (New England Biolabs, catalogue #E6040L). Total ChIP DNA (approximately 200-500ng) and 220ng of DNA input (WCE) was end repaired for 30 minutes at room temperature, and then purified using column purification with either DNA Clean and Concentrator (Zymogen, catalogue #D4014) or PCR purification columns (Qiagen, catalogue #28106) as recommended by manufacturer’s protocol. Blunt-end repaired DNA was dA-tailed for 40 minutes at 37° C, then column purified. dA-tailed DNA was ligated to Illumina adaptors (final concentration 6.67 nM) that have a T- overhang. USER enzyme was used to cleave the uracil hairpin of the Illumina adaptor, and adaptor-ligated DNA was column purified. The library was PCR amplified for 16-18 cycles using a universal primer and a barcoded primer (New England Biolabs, catalogue #E7335L). PCR-amplified DNA was purified using PCR column purification and eluted in 20 uL of elution buffer for preparation of gel extraction, or 30 uL of TE for preparation of Pippin Prep size selection. Library samples were size selected from 200-350bp using a 2% agarose dye-free automated size selection cassette from Pippin Prep (Sage Sciences, catalogue #CDF2010).