|
| Status |
Public on Jul 10, 2014 |
| Title |
U2OS_pSer5_Pol2_+Dox_ChIPseq |
| Sample type |
SRA |
| |
|
| Source name |
U2OS cells, +Dox, pSer5 Pol II ChIP
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: U2OS
doxycycline induction: +
chip antibody: anti-Pol II pSer5 (4H8) [Covance]
|
| Treatment protocol |
U2OS cells were induced with doxycycline (1µg/ml) for 30h.
|
| Growth protocol |
U2OS cells were grown in DMEM (Sigma) supplemented with 10% fetal calf serum (PAA) and penicillin/streptomycin (PAA).
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
For ChIP-seq, cells were crosslinked with 1% formaldehyde for 10' at 37°C. After cell lysis, nuclei were resuspended in RIPA buffer and sonicated (Bransson) until average fragment size was below 500 bps. Antibodies were bound to Protein-A/G-sepharose or Dyna beads and incubated with the chromatin. After sequential washing and elution with 1% SDS, crosslinking was reverted and DNA was purified using Qiagen PCR purification columns.
Libraries were constructed following manufacturer's instructions (NEBNext ChIP-Seq Sample Prep Kit). Briefly, ChIP DNA was end repaired, A tailed and Illumina adaptors were ligated. DNA fragments of about 200 bps were cut out of an agarose gel and extracted with a Qiagen PCR purification column. Afterwards, DNA was enriched with 18 PCR cycles, fragment size was controlled with Biorad Experion system and quantified using picogreen assay. Libraries were sequenced on an Illumina Genome Analyzer IIx following the manufacturer's instructions.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina Genome Analyzer IIx |
| |
|
| Data processing |
Basecalling was performed with the RTA package within the Genome Analyzer Data Collection Software (SCS2.8).
Fastq files were generated by CASAVA only considering high quality sequences (PF-cluster).
Overall sequencing quality was checked with the FastQC script.
Reads were aligned to the human genome with BOWTIE v0.12.7 or v0.12.8 using default parameters.
Peak calling was performed with MACS v1.4.2 with default parameters, but: model fold 25 (HeLa-Myc) and 15 (HeLa-Miz1), duplicates 3 (U2OS-Myc) or 10 (U2OS-Miz1).
Genome_build: GRCh37 (hg19)
Supplementary_files_format_and_content: .txt files from ChIP-seq experiments contain peak localisation in a MACS output -bed format.
|
| |
|
| Submission date |
Sep 13, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Martin Eilers |
| Organization name |
University of Wuerzburg
|
| Department |
Chair for Biochemistry and Molecular Biology
|
| Lab |
Martin Eilers
|
| Street address |
Am Hubland
|
| City |
Wuerzburg |
| ZIP/Postal code |
97074 |
| Country |
Germany |
| |
|
| Platform ID |
GPL10999 |
| Series (1) |
| GSE44672 |
Activation and repression by oncogenic Myc shapes tumour-specific gene expression profiles |
|
| Relations |
| BioSample |
SAMN02356451 |
| SRA |
SRX351406 |
