GSM3630249: H1 END FOXA2 gRNA ATACseq rep2; Homo sapiens; ATAC-seq
Sample information curated by ChIP-Atlas
Antigen
Antigen Class
ATAC-Seq
Antigen
ATAC-Seq
Cell type
Cell type Class
Pluripotent stem cell
Cell type
hESC derived endodermal cells
NA
NA
Attributes by original data submitter
Sample
source_name
Definitive endoderm
cell_line
H1
assay
ATAC
treatment protocol
FOXA2 gRNA
chip antibody
none
Sequenced DNA Library
library_strategy
ATAC-seq
library_source
GENOMIC
library_selection
other
library_construction_protocol
ATAC-seq samples were processed according to Buenrostro et. al with slight modifications. Briefly, approximately 50,000 cells were pelleted at 750 x g for 15 minutes at 4oC. After the addition of lysis buffer (10 mM Tris-Cl pH 7.4, 10 mM NaCl, 3 mM MgCl2, and 0.1% IGEPAl CA-630), nuclei were pelleted at 750 x g for 15 minutes at 4oC and the supernatent was discarded. Transposase reaction was performed at 37oC for 30 minutes. The purified, tagmented DNA was amplified for 9-10 cycles and size selected using AMPure XP beads (Beckman Coulter, A63881) as follows: 0.55X volume of 2X concentrated AMPure beads was added to the amplified DNA and incubated for 5 minutes at room temperature. The supernatant, containing low-molecular weight DNA, was collected in a separate tube by removing the beads using a magnetic rack. 1X volume of AMPure beads was added to the supernatant and incubated for 5 min at room temperature. The beads were washed twice with 75% ethanol and then air dried. The final purified library was eluted in EB buffer and sequenced (paired-end, 75-75) on a NextSeq500 or Hi-Seq2000 platform. Total RNA was isolated using Trizol Reagent (Invitrogen, 15596-018) according to the manufacturer's instructions. For quantitative PCR analysis, 1 μg of total RNA was reverse-transcribed using SuperScript III First-Strand Synthesis System (Thermo, 18080051). Bulk RNA-sequencing library preparation of H1 hESC and differentiated hEND was performed following a published protocol {Zhang:2012kc}. Briefly, 4 μg of total RNA was depleted of ribosomal RNA using Ribo-Zero rRNA Removal Kit (Epicentre, MRZH116) and then converted to cDNA. Final sequencing libraries were generated using dUTP (Thermo, R0133) incorporation and uracil-N-glycosylase (NEB, M0280) treatment to generate strand-specific RNA-seq libraries. The final libraries were sequenced at paired-end on a Hi-Seq2000 platform.