|
| Status |
Public on Jan 03, 2021 |
| Title |
3'-seq siRRP40xsiINTS11 xPAP rep 3 |
| Sample type |
SRA |
| |
|
| Source name |
tissue culture cells
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: HeLa Kyoto
tissue: cervix
cell type: epithelial
sirna: RRP40 + INTS11
rna treatment: polydenylation with E.coli poly(A) polymerase ('xPAP')
|
| Treatment protocol |
Two rounds of transfections were performed. The first transfections were performed using SilentFect (final dilution 1:1000) in RPMI 1640 medium. Two days after the first transfection, the media was changed, and the transfection was repeated using Lipofectamine 2000 (final dilution 1:1000) in RPMI 1640 medium. siRNAs were used at a final concentration of 15 nM of each siRNA. 24 hours after the second round of transfections, cells were labelled for 5 min or 10 min for TT-seq or 3'-seq, respectively, with 500 µM 4-thiouridine minutes and harvested.
|
| Growth protocol |
HeLa cells were cultured in standard DMEM medium supplemented with 10% FBS and penicillin-streptomycin mix.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
TT-seq/RNA-seq: Six in vitro transcribed spike-in RNAs were added to the lysates (TRIzol, Thermo) followed by extraction of RNA according to the manufacturer’s protocol. The RNA was then sonicated and used as input for RNA-seq or for further purification of 4sU-labeled RNA. 4sU-labeled RNA was first biotinylated with EZ-Link HPDP-Biotin (Pierce) and isolated with µMACS Streptavidin Kit (Miltenyi). 4sU-labeld RNA was eluted by cleaving off the biotin with DTT. Both total RNA and 4sU-labeled RNA purified prior to libary preparation using the miRNeasy Micro Kit (Qiagen) including DNse I treatment to remove genomic DNA.
3'-seq:
TT-seq/RNA-seq RNA libraries were prepared for sequencing using standard Illumina protocols (TruSeq Stranded Total RNA Sample Prep kit).
3'-seq libraries were generated with standard and modified Lexogen 3'-seq protocols. The modified preapration included 3'end polydenylation in vitro using E.coli poly(A) polymerase ('xPAP') preceeding the NGS library preparation with the QuantSeq REV kit according to manufacturer's protocol (Lexogen).
RNA-Seq, paired-end; RNA 3'end seq, single-end
|
| |
|
| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Description |
4sU-labeled RNA
|
| Data processing |
Illumina software used for basecalling (Casava) and demultiplexing
TT-seq/RNA-seq: the fastq files were mapped to the reference genome (GRCh38 + RNA spike-ins) using STAR aligner to generate the BAM alignment files
TT-seq/RNA-seq: the BAM files were filtered to contain only uniquely mapped reads with Samtools
TT-seq/RNA-seq: Size-factors for sequencing depth normalization were obtained using the DESeq2 algorithm from counts on reduced GenCode v21 annotation generated with HTSeq. The reads mapping to spike-ins were used to estimate antisense and contamination bias (contaminating unlabeled RNA in 4sU-labeledRNA). TT- and RNA-seq data were corrected accordingly. The experiments were done in duplicates in two batches and the data were batch corrected using the limma package.
TT-seq/RNA-seq: TT-seq/RNA-seq: Size-factors for sequencing depth normalization were obtained using the DESeq2 algorithm from counts on reduced GenCode v21 annotation generated with HTSeq. The reads mapping to spike-ins were used to estimate antisense and contamination bias (contaminating unlabeled RNA in 4sU-labeledRNA). TT- and RNA-seq data were corrected accordingly. The experiments were done in duplicates in two batches and the data were batch corrected using the limma package.
3'-seq: reads were trimmed using the bbduk.sh script from BBMAP and homopolymeric A-stretches removed from reads.
3'-seq: the data was mapped using STAR against a compound index composed of hg38 merged with ERCC spike-ins.
3'-seq: 5' ends of uniquely mapped reads were used for downstream analysis.
Genome_build: GRCh38
Supplementary_files_format_and_content: bigWig; normalized and bias corrected coverage generated with…
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| |
|
| Submission date |
Jun 05, 2020 |
| Last update date |
Jan 04, 2021 |
| Contact name |
Søren Lykke-Andersen |
| E-mail(s) |
[email protected]
|
| Phone |
50510996
|
| Organization name |
Aarhus University
|
| Department |
Molecular Biology and Genetics
|
| Street address |
C.F.Møllers Allé 3
|
| City |
Aarhus C |
| ZIP/Postal code |
8000 |
| Country |
Denmark |
| |
|
| Platform ID |
GPL18573 |
| Series (1) |
| GSE151919 |
Integrator is a genome-wide attenuator of non-productive transcription |
|
| Relations |
| BioSample |
SAMN15148798 |
| SRA |
SRX8483395 |
