|
| Status |
Public on May 08, 2018 |
| Title |
siBRMsiNS dex replicate 1 |
| Sample type |
SRA |
| |
|
| Source name |
U2OS-GR⍺ cells
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: U2OS cell line stably transfected with GRalpha
|
| Treatment protocol |
siRNA transfection was performed using Lipofractamine RNAimax (invitrogen) according to the manufacturer's protocol. 48 h after siRNA transfection the cells were treated for 1 hour with 100 nM dex (Sigma).
|
| Growth protocol |
Cells were grown in low glucose DMEM medium (ThermoFisher) supplemented with 5% (vol/vol) FBS
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
U2OS-GRα cells grown on 15-cm dishes were transfected with the appropriate siRNAs. After 48 h, the cells were treated with 100 nM dex for 1 h before crosslinking with 1% (v/v) formaldehyde for 10 mins at room temperature and extracting chromatin from the harvested cells. Chromatin was sonicated 20-30 min (30s on/off cycles) with the Biorupter (Diagenode) at 4ºC to produce DNA fragment size of 400-600 bp. Immunoprecipitation of the sonicated chromatin samples was conducted with a cocktail of GR antibodies: H300 (6 µg, Santa Cruz Biotechnology), PA1-511A (2 µg, Thermo Scientific), D6H2L (2 µg, Cell Signaling Technology) in a volume of 1 ml containing chromatin from 20 x 106 cells. Protein G Sepharose magnetic beads (GE Healthcare) were used to isolate the immune complexes with the crosslinked DNA.
Quality of the DNA was assessed using Agilent Technologies 2100 Bioanalyzer and RT-qPCR analysis was performed for selected GR binding sites as quality control. Standard illumina protocols were used to construct the library for sequencing.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Description |
GR_ChIP_06
|
| Data processing |
NextSeq Control Software (NCS) v1.4 was used for base calling
After trimming the raw reads for quality and adapter sequence, the samples were mapped using BWA with default parameters against the GRCh38/hg38 human reference genome
Duplicate reads and reads aligning to the mitochondrial DNA were discarded
The remaining mapped reads were quantified and peaks were called using MACS2 and IDR framework was used to determine the GR peaks for each condition
MACS+IDR peaks were called using Model-based Analysis for ChIP-Seq version 2 (MACS2) (22) and the Irreproducibility Discovery Rate (IDR) framework
Differentially bound peaks were identified using MACS2 called peaks followed by DiffBind analysis
Genome_build: hg38
Supplementary_files_format_and_content: comma separated file (csv) containing CPM values with each gene represented in each row and the experimental conditions as columns
|
| |
|
| Submission date |
Jan 18, 2018 |
| Last update date |
May 08, 2018 |
| Contact name |
Brian Hae Kang Lee |
| E-mail(s) |
[email protected]
|
| Organization name |
University of Southern California
|
| Department |
Biochemistry and Molecular Medicine
|
| Lab |
Michael R. Stallcup
|
| Street address |
1441 Eastlake Avenue, NOR 6316
|
| City |
Los Angeles |
| State/province |
CA |
| ZIP/Postal code |
90089 |
| Country |
USA |
| |
|
| Platform ID |
GPL18573 |
| Series (2) |
| GSE109383 |
Different Chromatin and DNA Sequence Characteristics Define Glucocorticoid Receptor Binding Sites that are Blocked or Not Blocked by Coregulator Hic-5 [ChIP-seq] |
| GSE109590 |
Different Chromatin and DNA Sequence Characteristics Define Glucocorticoid Receptor Binding Sites that are Blocked or Not Blocked by Coregulator Hic-5 |
|
| Relations |
| BioSample |
SAMN08377162 |
| SRA |
SRX3586572 |
