Sample information curated by ChIP-Atlas

Antigen

Antigen Class
TFs and others
Antigen
RPA1

Cell type

Cell type Class
Uterus
Cell type
HeLa
Primary Tissue
Cervix
Tissue Diagnosis
Adenocarcinoma

Attributes by original data submitter

Sample

source_name
RPA1 ChIP
cell line
Hela
medium
DMEM
cell cycle stage
asynchronization
chip antibody
RPA1 (gifted by Dr. Bruce Stillman)

Sequenced DNA Library

library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
HeLa cells were crosslinked with 2mM DSG in PBS for 30 min at room temperature, followed by treatment with 1% formaldehyde for 15 min. After quenching with glycine, the cells were harvested and digested by Mnase (NEB, M0247S,0.5u/1000 cells) at 37°c for 20mins to produce soluble chromatin with DNA fragments in the range of 150–300 bp. This chromatin was immunoprecipitated using antibodies against RPA1 or RPA2 overnight. Then, chromatin complexes were incubated with protein G beads for 3 h. After extensive washing steps, the DNA was recovered from the protein G complex using elution buffer (10 mM Tris at pH 8.0, 10 mM EDTA at pH 8.0, 1% SDS, 150 mM NaCL, 5 mM DTT). DNA was subsequently purified using Qiagen Mini Elute PCR purification kit. For RNA-seq, total RNA was extracted using the miRNeasy Mini kit (Qiagen, Valencia, CA). For Bru-seq, bromouridine labeling, isolation of Bru-RNA and strand specific cDNA library preparation were performed as described previously (Paulsen et al., 2014; Paulsen et al., 2013). Briefly, Bromouridine (Aldrich) was added to the media of HeLa cell to a final concentration of 2 mM, and cells were incubated at 37 °C for 30 min. Cells were then directly lysed by Trizol and isolated total RNA. Then Bru-labeled RNA was isolated from the total RNA by incubation with anti-BrdU antibodies (BD Biosciences) conjugated to magnetic beads under gentle agitation at room temperature for 1 h. ChIP-seq libraries were prepared from 10 ng ChIP and input DNA using the Ovation ultralow DR Multiplex kit (NuGEN, San Carlos, CA). The ChIP-seq libraries were sequenced to 51 base pairs from both ends on an Illumina HiSeq 2000 instrument in the Mayo Clinic Center for Individualized Medicine Medical Genomics Facility.RNA-seq libraries were prepared with ovation RNA-seq system v2 kit (NuGEN) according to the manufacture’s instruction, and were sequenced on an Illumina HiSeq 2000 instrument in the Mayo Clinic Center for Individualized Medicine Medical Genomics Facility. For Bru-seq, Isolated Bru-labeled RNA was used to prepare strand-specific DNA libraries by using the Illumina Tru Seq Kit (Illumina) according to the manufacturer’s instructions with modifications described as (Paulsen et al., 2014).

Sequencing Platform

instrument_model
Illumina HiSeq 2000

hg19

Number of total reads
37424391
Reads aligned (%)
173.2
Duplicates removed (%)
7.8
Number of peaks
213 (qval < 1E-05)

hg38

Number of total reads
37424391
Reads aligned (%)
174.3
Duplicates removed (%)
7.7
Number of peaks
281 (qval < 1E-05)

Base call quality data from DBCLS SRA