|
| Status |
Public on Jul 11, 2016 |
| Title |
ChIPseq_input_siCTR_siMYC |
| Sample type |
SRA |
| |
|
| Source name |
U2OS cells
|
| Organism |
Homo sapiens |
| Characteristics |
antibody: none
sirna treatment: siCTR and siMYC
doxycycline [ng/µl]: none
|
| Growth protocol |
U2OS cells were grown in DMEM (Sigma) suppelmented with 10% FCS (Biochrom) and penicillin/streptomycin (Sigma).
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
For ChIP-seq in MYC-induced U2OS cells, libraries from GEO record GSE44672 were used and sequenced deeper. For ChIP-seq in MYC-depleted U2OS cells, 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 Dyna beads and incubated with the chromatin. After sequential washing and elution with 1% SDS, crosslinking was reverted and DNA was purified using phenol-chloroform extraction and ethanol precipitation. 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 |
ChIPseq:
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 o the human genome usingBowtie v0.12.8. with default settings. For siRNA-treated samples, reads were trimmed 5bp from the 3' end.
Samples were normalized to the same number of aligned reads.
Bam files were generated using Samtools v0.1.18.
For the generation of wiggle files MACS v1.4.2 was used with a fixed step of 10bp and the "keep-dup" parameter was set to 3.
Genome_build: hg19
Supplementary_files_format_and_content: fixedStep wiggle files
|
| |
|
| Submission date |
Apr 23, 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) |
| GSE77356 |
Different promoter affinities account for specificity in MYC-dependent gene regulation |
|
| Relations |
| BioSample |
SAMN04889574 |
| SRA |
SRX1722158 |
