GSM6940146: E135 RAD21, CTCF, BioRep2; Homo sapiens; ChIP-Seq
Sample information curated by ChIP-Atlas
Antigen
Antigen Class
TFs and others
Antigen
RAD21
Cell type
Cell type Class
Pluripotent stem cell
Cell type
hESC derived neural crests
NA
NA
Attributes by original data submitter
Sample
source_name
H9 hESC
cell line
H9 hESC
cell type
hESC-derived P4 CNCC
genotype
SSE1.35 CTCF site deletion
chip antibody
RAD21 (Abcam, #ab992)
Sequenced DNA Library
library_name
GSM6940146
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
5-15 million cells were cross-linked per ChIP experiment in 2mL PBS per 6-well with 1% methanol-free formaldehyde for 5-10 min and quenched with a final concentration of 0.125M glycine for 5 min with nutation. Cross-linked cells were washed with PBS, scraped and pelleted by centrifugation, flash-frozen in liquid nitrogen and stored at -80°C. Samples were defrosted on ice and resuspended in 5mL LB1 (50 mM HEPES-KOH pH 7.5, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP-40, 0.25% Triton X-100, with 1X cOmplete Protease Inhibitor Cocktail and 1 mM PMSF) and rotated vertically for 10 min at 4°C. Samples were centrifuged for 5 min at 1350 x g at 4°C, and resuspended in 5mL LB2 (10 mM Tris, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, with 1X cOmplete Protease Inhibitor Cocktail and 1mM PMSF) and rotated vertically for 10 min at 4°C. Samples were centrifuged for 5 min at 1350 x g at 4°C, and resuspended in 1mL LB3 per 10 million cells (up to 1 mL per ChIP). Samples were sonicated in 1mL AFA tubes for 5 min on E220 evolution Covaris with settings Peak power = 140, Duty Factor = 10, Cycles per burst = 200 to achieve chromatin sized approximately 500-2000bp. Following sonication, samples were re-combined (if aliquoted for sonication), Triton X-100 was added to the fragmented chromatin to a final concentration of 1%, and the chromatin divided for input (1%–2%) and ChIP samples. Samples were quantified by Qubit dsDNA HS assay kit, and 30 ng of ChIP DNA was used for library preparation with end repair, A-tailing, and adaptor ligation (NEB). Following USER enzyme treatment, libraries were cleaned up with one round of single-side AMPure XP bead clean-up. Libraries were then amplified to add indices using NEBNext HiFi 2X PCR mix and NEBNext Multiplex Oligos for Illumina kit (NEB, E7335S) with 9 cycles (as determined by qPCR. ChIP libraries were purified by two rounds of double-sided AMPure XP bead clean-up to deplete adaptors. Library concentration and quality within ChIP or input groups was assessed by Qubit dsDNA HS assay kit and used to pool within ChIP or input groups.