GSM5932289: ChIPseq 2AKO GRHL2 H9 d7 rep2; Homo sapiens; ChIP-Seq
Sample information curated by ChIP-Atlas
Antigen
Antigen Class
TFs and others
Antigen
GRHL2
Cell type
Cell type Class
Pluripotent stem cell
Cell type
hESC H9
NA
NA
Attributes by original data submitter
Sample
source_name
H9 day7
treatment
7 days RA/BMP4
chip antibody
GRHL2, Sigma, AB_1857928
Sequenced DNA Library
library_name
GSM5932289
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
Cells were grown on 15-cm dishes (25 million cells per replicate), singularized by Accutase (Innovative Cell Technologies), crosslinked by 1% formaldehyde for 10 minutes, lysed (50mM Tris-HCl pH 8.0, 10mM EDTA, 0.5% SDS, 1X protease inhibitors), and sonicated to 200-300 bp size using a Bioeruptor (Diagenode). Samples were centrifuged to remove insoluble debris and diluted in dilution buffer (50 mM Tris-HCl pH 8.0, 10mM EDTA, 1X protease inhibitors) to final concentration of 0.1% SDS. Sheared chromatin was incubated overnight at 4°C with appropriate antibodies, followed by incubation on rotator with 30μL of pre-washed agarose G beads (Invitrogen) for 4h at 4°C. AP2A ChIP antibody (Santa Cruz: sc-12726) used at 20μg per 25 million cells and GRHL2 ChIP antibody (Sigma Prestige: HPA004820) used at 6μg per 25 million cells. Beads were washed twice each with low salt buffer (50 mM Tris-HCl pH 8.0, 0.15M NaCl, 1mM EDTA pH 8.0, 0.1% SDS, 1% triton X-100, 0.1% sodium deoxycholate), high salt buffer (50mM Tris-HCl pH 8.0, 0.5M NaCl, 1mM EDTA pH 8.0, 0.1% SDS, 1% triton X-100, 0.1% sodium deoxycholate), and LiCl buffer (50mM Tris-HCl pH 8.0, 0.15M LiCl, 1mM EDTA pH 8.0, 1% NP-40, 0.1% sodium deoxycholate). DNA was eluted in 100μL of elution buffer (50mM NaHCO3, 1% SDS) and crosslinks were reversed with 4μL of 5M NaCl incubated overnight at 67°C. RNA was removed by adding 1μL of 10 mg/mL RNase A and incubating for 30 min at 37°C. DNA was cleaned using the Qiagen Qiaquick PCR purification kit and quantified using Qubit (Invitrogen). Between 5ng and 1μg of pooled DNA were used for library preparation using NEBNext Ultra™ II DNA Library Prep Kit for Illumina kit (New England Biolabs) and AMPure XP beads (Beckman) according to the manufacturer's protocol. Single-read libraries were sequenced on Illumina NextSeq or NovaSeq sequencer. Two independent, biological replicates were sequenced per cell type.