Sample information curated by ChIP-Atlas

Antigen

Antigen Class
TFs and others
Antigen
BRD3

Cell type

Cell type Class
Bone
Cell type
U2OS
Tissue
bone
Lineage
mesoderm
Description
osteosarcoma from the tibia of a 15 year old, J. Ponten and E. Saksela derived this line (originally 2T) in 1964 from a moderately differentiated sarcoma, viruses were not detected during co-cultivation with WI-38 cells or in CF tests against SV40, RSV or adenoviruses, mycoplasma contamination was detected and eliminated in 1972, (PMID: 6081590)

Attributes by original data submitter

Sample

Alias
E-MTAB-5670:BRD3-WT1
Broker name
ArrayExpress
Description
Protocols: Flp-In T-REx U-2 OS cells stably expressing GFP tagged BRD3 WT or a N116A N391A construct (bd(1+2)) were grown in 2 x 15 cm plates to ~75% confluence and induced with 1 µg/mL tetracycline for 48 hours. Cells were then washed with PBS and crosslink with 1% formaldehyde in PBS for 15 minutes at room temperature before quenching the reaction with 125 mM glycine in PBS for 5 minutes at room temperature. Cells were then pelleted and frozen at -80°C until ChIP was performed. Chromatin immunoprecipitation (ChIP) was performed as described previously (REF, PMID:19275939) using 7 µg of anti-GFP (ab290, Abcam) antibody. 220 ng of input control DNA extracted from sonicated cell lysates of each sample were processed in parallel. ChIP DNA was prepared for Illumina sequencing by blunt-end repair, dA-tailing, and ligation of Illumina adaptors using a NEBNext DNA library preparation kit (New England Biolabs, catalogue #E6040L). Total ChIP DNA (approximately 200-500ng) and 220ng of DNA input (WCE) was end repaired for 30 minutes at room temperature, and then purified using column purification with either DNA Clean and Concentrator (Zymogen, catalogue #D4014) or PCR purification columns (Qiagen, catalogue #28106) as recommended by manufacturer’s protocol. Blunt-end repaired DNA was dA-tailed for 40 minutes at 37° C, then column purified. dA-tailed DNA was ligated to Illumina adaptors (final concentration 6.67 nM) that have a T- overhang. USER enzyme was used to cleave the uracil hairpin of the Illumina adaptor, and adaptor-ligated DNA was column purified. The library was PCR amplified for 16-18 cycles using a universal primer and a barcoded primer (New England Biolabs, catalogue #E7335L). PCR-amplified DNA was purified using PCR column purification and eluted in 20 uL of elution buffer for preparation of gel extraction, or 30 uL of TE for preparation of Pippin Prep size selection. Library samples were size selected from 200-350bp using a 2% agarose dye-free automated size selection cassette from Pippin Prep (Sage Sciences, catalogue #CDF2010).
ENA checklist
ERC000011
INSDC center alias
SickKids Research Institute Department of Molecular Genetics, University of Toronto
INSDC center name
SickKids Research Institute Department of Molecular Genetics, University of Toronto
INSDC first public
2018-04-01T17:02:53Z
INSDC last update
2017-04-11T13:29:20Z
INSDC status
public
SRA accession
ERS1663585
Sample Name
ERS1663585
Title
BRD3-WT1
cell_line
U-2 OS cell
cell_type
osteosarcoma cell
genotype
GFP-BRD3
organism
Homo sapiens
sex
female

Sequenced DNA Library

library_name
BRD3-WT1_s
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
Flp-In T-REx U-2 OS cells stably expressing GFP tagged BRD3 WT or a N116A N391A construct (bd(1+2)) were grown in 2 x 15 cm plates to ~75% confluence and induced with 1 µg/mL tetracycline for 48 hours. Cells were then washed with PBS and crosslink with 1% formaldehyde in PBS for 15 minutes at room temperature before quenching the reaction with 125 mM glycine in PBS for 5 minutes at room temperature. Cells were then pelleted and frozen at -80°C until ChIP was performed. Chromatin immunoprecipitation (ChIP) was performed as described previously (REF, PMID:19275939) using 7 µg of anti-GFP (ab290, Abcam) antibody. 220 ng of input control DNA extracted from sonicated cell lysates of each sample were processed in parallel. ChIP DNA was prepared for Illumina sequencing by blunt-end repair, dA-tailing, and ligation of Illumina adaptors using a NEBNext DNA library preparation kit (New England Biolabs, catalogue #E6040L). Total ChIP DNA (approximately 200-500ng) and 220ng of DNA input (WCE) was end repaired for 30 minutes at room temperature, and then purified using column purification with either DNA Clean and Concentrator (Zymogen, catalogue #D4014) or PCR purification columns (Qiagen, catalogue #28106) as recommended by manufacturer's protocol. Blunt-end repaired DNA was dA-tailed for 40 minutes at 37° C, then column purified. dA-tailed DNA was ligated to Illumina adaptors (final concentration 6.67 nM) that have a T- overhang. USER enzyme was used to cleave the uracil hairpin of the Illumina adaptor, and adaptor-ligated DNA was column purified. The library was PCR amplified for 16-18 cycles using a universal primer and a barcoded primer (New England Biolabs, catalogue #E7335L). PCR-amplified DNA was purified using PCR column purification and eluted in 20 uL of elution buffer for preparation of gel extraction, or 30 uL of TE for preparation of Pippin Prep size selection. Library samples were size selected from 200-350bp using a 2% agarose dye-free automated size selection cassette from Pippin Prep (Sage Sciences, catalogue #CDF2010).

Sequencing Platform

instrument_model
Illumina HiSeq 2500

hg19

Number of total reads
21079768
Reads aligned (%)
95.8
Duplicates removed (%)
4.8
Number of peaks
3868 (qval < 1E-05)

hg38

Number of total reads
21079768
Reads aligned (%)
96.3
Duplicates removed (%)
3.7
Number of peaks
4003 (qval < 1E-05)

Base call quality data from DBCLS SRA