title: WL377
organism: Homo sapiens
description: Protocols: 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).
attributes.tag: ENA first public
attributes.value: 2017-06-15
attributes.tag: ENA last update
attributes.value: 2016-10-17
attributes.tag: External Id
attributes.value: SAMEA4515522
attributes.tag: INSDC center alias
attributes.value: Scientist, Genetics & Genome Biology Program, SickKids Research Institute Assistant Professor, Department of Molecular Genetics, University of Toronto
attributes.tag: INSDC center name
attributes.value: Scientist, Genetics & Genome Biology Program, SickKids Research Institute Assistant Professor, Department of Molecular Genetics, University of Toronto
attributes.tag: INSDC first public
attributes.value: 2017-06-15T17:02:09Z
attributes.tag: INSDC last update
attributes.value: 2016-10-17T14:21:08Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-5148:WL377
attributes.tag: antibody part number
attributes.value: 05-1334
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell type
attributes.value: HUVEC cell
attributes.tag: common name
attributes.value: human
attributes.tag: individual
attributes.value: 12508
attributes.tag: sample name
attributes.value: E-MTAB-5148:WL377