|
| Status |
Public on Dec 10, 2021 |
| Title |
4sU_4-OHT.merged |
| Sample type |
SRA |
| |
|
| Source name |
SH-EP-MYCNER
|
| Organism |
Homo sapiens |
| Characteristics |
treatment (etoh or 4-oht): 4-OHT (200 nM)
treatment (etoh ordox): N/A
treatment (dmso or ku-60019): DMSO
label: 20min 4sU
antibody: N/A
|
| 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 |
total RNA |
| Extraction protocol |
SH-EP-MYCNER cell lysates were spiked with 4sU-treated mouse T-cell lysates for the no chase 4sU. Total RNA was isolated using QIAzol and purified with miRNeasy columns and biotinylated. This was followed by 4sU-RNA pulldown pulldown using streptavidin-MyOne beads, followed by purification with RNA minElute columns (Qiagen)
4sU-RNA was used for library preparation with NEBNext rRNA Depletion Kit (Human/Mouse/Rat) and NEBNext Ultra II Directional RNA Library Prep kit. The libraries were amplified with 10-14 PCR cycles depending on the input RNA. The concentration and size distribution of the libraries were determined on a Fragment Analyzer using the NGS Fragment High Sensitivity Analysis Kit (1-6,000 bp; Agilent Technologies). The libraries were sequenced on the NextSeq500 Illumina platform for 75 cycles. Base calling was performed using Illumina’s BaseSpace platform.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Description |
nascent RNA
|
| 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 |
SAMN17283614 |
| SRA |
SRX9821815 |
