 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Aug 07, 2018 |
| Title |
mcf7_ctbp2 |
| Sample type |
SRA |
| |
|
| Source name |
MCF7
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: MCF7
cell type: breast cancer cell line
genotype/variation: WT
yap 5sa induced: No
chip antibody: CtBP2 (BD Bioscience, Cat# 612044)
|
| Treatment protocol |
For YAP ChIP, an MCF7 cell line carrying a doxycycline-inducible YAP 5SA allele was used. YAP 5SA was induced by adding 1 µg/ml doxycycline for 18 hours and subsequently subjected to formaldehyde crosslinking. For generating TRPS1 KO cells by CRISPR, MCF7 cells were transfected with a pX461 vector (carrying WT Cas9 and not the nickase version) carrying a sgRNA sequence that targets human TRPS1. Transfection was performed using Lipofectamine 3000. Transfected (GFP+) cells were sorted by flow cytometry into a 96-well plate as single cells. The Knockout of TRPS1 was verified by Western Blot and Sanger Sequencing.
|
| Growth protocol |
MCF7 and T47D cells were cultured in DMEM (+GlutaMAX, Thermo Scientific) supplemented with 10% FBS (Sigma) and 1% penicillin-streptomycin (Sigma). T47D culture medium was additionally supplemented with 1 µl/ml human insulin solution (Sigma).
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were cross-linked with 1% formaldehyde for 10 min at RT. Nuclei were extracted using hypotonic buffer (20 mM Tris, pH 7.4, 2 mM MgCl2, 5% glycerol) and lysed with ChIP lysis buffer (20 mM Tris pH 7.4, 150 mM NaCl, 1% NP40, 0.5% DOC, 0.1% SDS, 1 mM EDTA). Chromatin was sonicated to obtain fragments of 200 bp. Equilibration of beads (Dynabeads, Thermo Scientific) with antibodies (2 μg for ChIP, 10 μg for ChIP-Seq) was performed overnight, following immunoprecipitation of chromatin for 6h. After extensive washing, bound chromatin was eluted using ChIP elution buffer (50 M Tris, pH 8.0, 1 mM EDTA, 1% SDS, 50 mM NaHCO3) and de-crosslinked and RNase A/Proteinase K (Roth) digested over night. DNA was purified by phenol/chloroform extraction and ethanol precipitation.
ChIP-Seq libraries were generated according to the manufacturer’s protocol (NEBNext Ultra II DNA Library Prep Kit for Illumina, NEB) using Dual Index Primers (NEBNext Multiplex Oligos for Illumina, NEB). The number of PCR cycles varied between 9-12 PCR cycles. The optimal PCR cycle number was determined beforehand using a qPCR machine with 10% of the material used for the final amplification.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Data processing |
FASTQ files were processed (adapter removal, length filter, quality filter) using cutadapt (version 1.12) with the following options: --discard-trimmed -m 25 -q 10
FASTQ files were aligned to hg19 using bowtie2 (version 2.2.9) using the --local option
Peak calling was performed using MACS2 (version 2.1.1) using the following settings: B --nomodel --SPMR -g hs --extsize 147. The resulting normalized (per million reads) bedgraph files were converted to bigWig files using bdg2bw.
Genome_build: hg19 (GRCh37)
Supplementary_files_format_and_content: *bw: The MACS2-derived normalized (per million reads) bedgraph files were converted to bigWig files using bdg2bw.
|
| |
|
| Submission date |
Nov 16, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Bjoern von Eyss |
| E-mail(s) |
bjoern.voneyss@leibniz-fli.de
|
| Organization name |
Leibniz Institute on Aging
|
| Lab |
von Eyss
|
| Street address |
Beutenbergstr. 11
|
| City |
Jena |
| ZIP/Postal code |
07745 |
| Country |
Germany |
| |
|
| Platform ID |
GPL16791 |
| Series (2) |
| GSE107013 |
TRPS1 shapes YAP/TEAD-dependent transcription in breast cancer cells [ChIP-seq] |
| GSE107023 |
TRPS1 shapes YAP/TEAD-dependent transcription in breast cancer cells |
|
| Relations |
| BioSample |
SAMN08035676 |
| SRA |
SRX3401276 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2859579_mcf7_ctbp2_treat_pileup.bw |
195.1 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
|
|
|
|
 |
