|
| Status |
Public on Feb 06, 2020 |
| Title |
siGFP_xPAP_in_batch2 |
| Sample type |
SRA |
| |
|
| Source name |
tissue culture cells
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: HeLa
sirna: siGFP
rna fraction: xPAP
pap treatment: in
batch: batch2
|
| Treatment protocol |
siRNA transfections were performed targeting EGFP(control), RBM7, RRP40, ZCCHC8, ZFC3H1 or ZCCHC8+ZFC3H1. Cells were transfected with siRNAs twice with the second time repeated after two days, and then cell were cultured for another two days. 10 min before harvesting, 4-thiouridine (4sU) was added to the growth medium for metabolic labelling of early RNA.
|
| Growth protocol |
HeLa cells were cultured in standard DMEM medium supplemented with 10% FBS and penicillin-streptomycin mix.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNAs were extracted using TRIzol (Invitrogen) according to the manufacturer’s protocol and then treated with TURBO DNase (Invitrogen) following the manufacturer’s instruction to remove contaminating genomic DNA. Six in vitro transcribed spike-in RNAs were added. Sequences for spike-ins are identical to ERCC spike-ins. ERCC-00092, ERCC-00136 and ERCC-00043 were transcribed in the presence of 4sU, whereas ERCC-00170, ERCC-00002 and ERCC-00145 were transcribed without 4sU and serve as negative control in the 4sU purification step.
An aliquot of each RNA was analyzed as total RNA, spiked with an additional full complement of commercially available ERCC spike-ins. The remainder of RNA was used for 4sU purification. Half of all total and 4sU labelled RNA samples were 3'end polydenylated in vitro using E.coli poly(A) polymerase ('xPAP'). All samples were rRNA depleted and subjected to pA+ RNA 3'end sequencing using QuantSeq REV kit from Lexogen GmbH. 8 or 10 libraries were multiplexed and sequenced at the Vienna Biocenter Core Facilities with a NextSeq SR75 High Output run.
RNA 3'end seq, single-end
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Data processing |
3'end seq reads were trimmed using the bbduk.sh script from BBMAP and homopolymeric A-stretches removed from reads.
The data was mapped using STAR against a compound index composed of hg38 merged with ERCC spike-ins.
5' ends of uniquely mapped reads were used for downstream analysis.
Genome_build: hg38
Supplementary_files_format_and_content: bigWig files were generated from bam files using a custom script. The score represents normalized counts per base.
|
| |
|
| Submission date |
Sep 17, 2019 |
| Last update date |
Feb 06, 2020 |
| Contact name |
Manfred Schmid |
| E-mail(s) |
ms@mbg.au.dk
|
| Organization name |
Aarhus University
|
| Department |
Molecular Biology and Genetics
|
| Lab |
Torben Heick Jensen
|
| Street address |
CF Møllers Alle bldg 1130
|
| City |
Aarhus C |
| ZIP/Postal code |
8000 |
| Country |
Denmark |
| |
|
| Platform ID |
GPL18573 |
| Series (1) |
| GSE137612 |
Distinct and redundant roles of nuclear RNA exosome targeting complexes |
|
| Relations |
| BioSample |
SAMN12776619 |
| SRA |
SRX6862474 |
