GSM4445332: HUVEC ETV2 Flag3 1; Homo sapiens; ChIP-Seq
Sample information curated by ChIP-Atlas
Antigen
Antigen Class
TFs and others
Antigen
Epitope tags
Cell type
Cell type Class
Cardiovascular
Cell type
HUVEC
Primary Tissue
Umbilical Cord
Tissue Diagnosis
Normal
Attributes by original data submitter
Sample
source_name
Human Umbilical Vein Endothelial Cells (HUVEC)
cell type
HUVEC
chip antibody
Flag (Sigma F1804)
treatment
lenti ETV2
growth
induction (flat 2d)
Sequenced DNA Library
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
To identify genome-wide localization of ETV2 in R-VEC, ChIP assays were performed with approximately 1x10e7 cells per experiment. Cells introduced with triple flagged ETV2 lentivirus were used for the ETV2 ChIP. Briefly, cells were crosslinked in 1% paraformaldehyde (PFA) for 10 min at 37°C, then quenched by 0.125M glycine. Chromatin was sheared using a Bioruptor (Diagenode) to create fragments of 200-400 bp, immunoprecipitated by 2–5 ug of antibody bound to 75 ml Dynabeads M-280 (Invitrogen) and incubated overnight at 4°C. Magnetic beads were washed and chromatin was eluted. The ChIP DNA was reverse-crosslinked and column-purified. To identify genome-wide localization of K4me3, K27me3, and K27ac modification, small-scale ChIP assays were performed with approximately 1x104 cells per experiment. Briefly, cells were washed once with nuclei extraction buffer (10 mM Tris-HCl pH 8.5, 2 mM EDTA, 140 mM NaCl, 5 mM MgCl2, 0.6% NP40, 1x EDTA-free protease inhibitor cocktail and 1 mM phenylmethanesulfonyl fluoride). The chromatin was fragmented for 5 min using MNase at 25 °C, diluted in ChIP buffer (10 mM Tris-HCl pH 8.0, 2 mM EDTA, 90 mM NaCl, 0.1% Triton X-100, 0.1% NaDOC, 1x EDTA-free protease inhibitor cocktail and 1 mM phenylmethanesulfonyl fluoride), and then immunoprecipitated with 1 ug of antibody bound to 10 ml Pierce Protein A/G Magnetic Beads (Invitrogen) and incubated overnight at 4°C. Magnetic beads were washed and chromatin was eluted. The ChIP DNA was reverse-crosslinked, purified by phenol chloroform, and precipitated by ethanol. ChIP samples were prepared for sequencing using KAPA Hyper Prep Kit.