 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Dec 10, 2021 |
| Title |
ChIPRX_pSer2RNAPII_shDCP1A_Dox_R2 |
| Sample type |
SRA |
| |
|
| Source name |
SH-EP-MYCNER shDCP1A
|
| Organism |
Homo sapiens |
| Characteristics |
treatment (etoh or 4-oht): EtOH
treatment (etoh ordox): Dox (1 µg/ml)
treatment (dmso or ku-60019): N/A
label: N/A
antibody: RNA polymerase II CTD repeat YSPTSPS (phospho S2), Abcam, ab5095
|
| Treatment protocol |
For shRNA experiments, cells were treated for 72 h with Dox (1 µg/ml) before adding 4-OHT (200 nM) for 4 h. For the ATM inhibition 4sU, cells were treated for 4 h with 4-OHT (200 nM) and/or KU-60019 ATM inhibitor (100 nM). For all 4sU experiments, 4sU (500 µM) was added in the last 20 min of treatment.
|
| Growth protocol |
SH-EP-MYCNER cells stably expressing Dox-inducible shRNAs against EXOSC10, TCEA1 or DCP1A were grown in RPMI-1640 (Thermo Fisher Scientific), supplemented with 10% fetal calf serum (Capricorn Scientific) and 1% penicillin/streptomycin (Sigma Aldrich).
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were treated with 1% formaldehyde for 5 min at 37°C. After cell lysis, nuclei were re-suspended in RIPA buffer (10 mM Tris/HCl pH 7.5, 150 mM NaCl, 1% NP40, 1% deoxycholic acid (DOC), 0.1% SDS, 1 mM EDTA) and DNA was fragmented to a size <300 bp using Covaris. Chromatin was immunoprecipitated with 15 µg of corresponding antibody for 6 hours. Chromatin was eluted with 1% SDS and crosslinking was reverted overnight. For purification, chloroform/phenol extraction was used.
Libraries were prepared according to instructions accompanying the NEBNext Ultra™ II DNA Library Prep Kit for Illumina. Purified DNA was end-repaired, A-tailed, ligated to Illumina adaptors, size-selected (200 bp) and eluted. DNA fragments were amplified by 12 to 17 cycles of PCR and library size was tested with the Fragment Analyzer (Advanced Analytical). The amount of library DNA was quantified using a picogreen assay
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Data processing |
Fastq files were generated Illumina’s BaseSpace platform and overall sequencing quality was analyzed with the FastQC script.
For ChIP-seq, fastq files were mapped to the human genome (hg19) using Bowtie v2.2.7. and normalized to the sample with the spike in control (mm10) except for MYCN ChIPseq, which was read normalized
For 4sU-seq, reads were mapped to hg19 with Bowtie2, reads mapping to rRNA,exons and blacklist regions were removed, and then normalized based on read depth, except for no chase 4sUseq, which was normalized to spike in control (mm10)
Genome_build: hg19
Supplementary_files_format_and_content: bedGraph files as defined by UCSC (https://genome.ucsc.edu/goldenpath/help/bedgraph.html).
|
| |
|
| Submission date |
Jan 11, 2021 |
| Last update date |
Dec 10, 2021 |
| 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 (2) |
| GSE164555 |
The MYCN oncoprotein resolves conflicts of stalling RNA Polymerase with the replication fork [DP_EXOSC10_ChIP_4sUseq] |
| GSE164569 |
The MYCN oncoprotein resolves conflicts of stalling RNA Polymerase with the replication fork |
|
| Relations |
| BioSample |
SAMN17283617 |
| SRA |
SRX9821812 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5014023_ChIPRX_pSer2RNAPII_shDCP1A_Dox_R2.spike.normalized.bedGraph.gz |
114.9 Mb |
(ftp)(http) |
BEDGRAPH |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
|
|
|
|
 |
