|
| Status |
Public on Apr 13, 2017 |
| Title |
EV_plusQ_input |
| Sample type |
SRA |
| |
|
| Source name |
HCT116
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: HCT116
treatment1(myc-er+oht or empty vector +oht): vector+OHT
treatment2(glutamine(q) concentration): 2mM
antibody: none
|
| Treatment protocol |
Cells were treated with 100 nM 4-Hydroxytamoxifen for 15 hours, then glutamine starved for 5 hours in presence of 4-Hydroxytamoxifen.
|
| Growth protocol |
HCT116 cells were maintained in DMEM supplemented with 10% FBS (Biochrom) and 1% penicillin/streptomycin. Experiments were performed using DMEM reconstituted with 2 mM glutamine, 2.5 g/l glucose, 0.015 g/l phenol red, 10% dialyzed FBS and 1% penicillin/streptomycin, without sodium pyruvate . For starvation experiments, cells were washed twice with PBS and cultured in reconstituted DMEM without glutamine.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
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 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 |
Fastq files were generated using Illumina's CASAVA software.
Reads were mapped to the human genome (hg19) using Bowtie v1.1.1 with default parameters and normalized to the sample with the smallest number of mapped reads.
Bedgraph files were generated with the genomeCoverageBed function from BedTools.
Genome_build: hg19
Supplementary_files_format_and_content: BedGraph format as described in the UCSC file format guide (https://genome.ucsc.edu/goldenpath/help/bedgraph.html).
|
| |
|
| Submission date |
Sep 07, 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) |
| GSE86556 |
The 3’-UTR of MYC couples RNA polymerase II function to ribonucleotide levels |
|
| Relations |
| BioSample |
SAMN05751909 |
| SRA |
SRX2148696 |
