|
| Status |
Public on Jan 30, 2023 |
| Title |
MCF10A_STAG2_CTCF KD_ChIP-seq |
| Sample type |
SRA |
| |
|
| Source name |
breast
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: breast
cell line: MCF10A
cell type: epithelial
chip antibody: STAG2, custom made
treatment: CTCF siRNA
|
| Treatment protocol |
MCF10A cells were transfected with 50 nM siRNAs using DharmaFECT reagent 1 and Gibco Opti-MEM I Reduced Serum Media and harvested 72h later.
|
| Growth protocol |
MCF10A cells are grown AT 37ºC at 5% CO2 in DMEM/F12 supplemented with 20ng/ml of EGF, 0.5mg/ml hydrocortisone, 100ng/ml of cholera toxin, 10mg/ml of insulin and 5% of horse serum.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were fixed for 15 min in 1% formaldehyde and after quenchig and lysis, sonication with performed in Covaris instrument. Around 50 micrograms of antibody was added to immunoprecipitate chromatin from 10 million cells. 5% mouse ES cell chromatin was added for calibration.
At least 5 ng of DNA were processed through subsequent enzymatic treatments with "NEBNext Ultra II FS DNA Library Prep Kit for Illumina" from New England BioLabs (ref. E7805). Briefly, a short fragmentation of 10 min was followed by end-repair, dA-tailing, and ligation to adapters. Adapter-ligated libraries were completed by limited-cycle PCR (8-12 cycles). Resulting average fragment size is 300 bp from which 120 bp correspond to adaptor sequences. Libraries were applied to an Illumina flow cell for cluster generation and sequenced on Illumina NextSeq 500 (with v2.5 reagent kits) following manufacturer's recommendations.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
NextSeq 550 |
| |
|
| Data processing |
Alignment of reads to the reference human genome (hg19) was performed using ‘Bowtie2′ (version 2.4.2) under default settings
Duplicates were removed using GATK4 (version 4.1.9.0)
peak calling was carried out using MACS2 (version 2.2.7.1) after setting the q value (FDR) to 0.05 and using the ‘–extsize’ argument with the values obtained in the ‘macs2 predictd’ step
Assembly: hg19
Supplementary files format and content: bigwig
|
| |
|
| Submission date |
Jun 28, 2022 |
| Last update date |
Jan 30, 2023 |
| Contact name |
Daniel Gimenez |
| E-mail(s) |
dgimenezl@cnio.es
|
| Organization name |
CNIO
|
| Street address |
Calle de Melchor Fernández Almagro, 3
|
| City |
Madrid |
| ZIP/Postal code |
28029 |
| Country |
Spain |
| |
|
| Platform ID |
GPL21697 |
| Series (2) |
| GSE207113 |
Response of cohesin STAG1 and STAG2 to reduction of NIPBL levels [ChIP-Seq] |
| GSE207116 |
Response of cohesin STAG1 and STAG2 to reduction of NIPBL levels |
|
| Relations |
| BioSample |
SAMN29402537 |
| SRA |
SRX15929738 |
