|
| Status |
Public on Oct 18, 2016 |
| Title |
U2OS_ChIPseq_vector_-Dox_IgG |
| Sample type |
SRA |
| |
|
| Source name |
U2OS cells
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: U2OS
treatment1(constitutive ha-omomyc or empty vector): vector
treatment2 (doxycycline to induce myc expression): Ethanol
antibody: IgG (rabbit, Sigma)
|
| Treatment protocol |
U2OS cells were treated with doxycycline (1µg/ml) for 30h (RNA-seq) or 8h (ChIP-seq) to induce MYC expression. For the shRNA screen, KPC cells were infected with doxycycline inducible lentiviral shRNA vectors and shRNA expression was induced with 1µg/ml doxycycline for 5 days.
|
| Growth protocol |
U2OS and KPC cells were grown in DMEM (Sigma) supplemented with 10% fetal calf serum (Biochrom) and penicillin/streptomycin (Sigma). The U2OS tet-on-MYC cell line was generated as described previously (Walz et al., 2014). For constitutive expression, N-terminally HA-tagged OmoMYC or various mutants (generated using site-directed mutagenesis) were cloned into SFFV-driven lentiviral expression vectors. Lentiviruses were produced by co-transfection with the packaging plasmid psPAX.2, and the envelope plasmid pMD2.G into HEK293 cells using the PEI method. Cells were infected with lentiviral supernatants in the presence of 4 mg/ml polybrene (Sigma) for 24 h and selected with 2 µg/ml Puromycin (InvivoGen).
|
| 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.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Data processing |
For ChIP-seq, fastq files were mapped to the human genome (hg19) using Bowtie v1.1.1. and normalized to the sample with the smallest number of mapped reads.
Wiggle files were generated using MACS v1.4.2 with default parameters but a p-value cutoff of 1e-6.
hg19
Fixed step wiggle files with a resolution of 10bp.
Fastq files were generated using Illumina's CASAVA software.
|
| |
|
| Submission date |
Jan 28, 2016 |
| 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 |
GPL18573 |
| Series (1) |
| GSE77328 |
OmoMYC blunts promoter invasion by oncogenic MYC to inhibit gene expression characteristic of MYC-dependent tumors |
|
| Relations |
| BioSample |
SAMN04445837 |
| SRA |
SRX1550248 |
