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| Status |
Public on Dec 16, 2022 |
| Title |
HEK293 Fast Pol II Nanopore |
| Sample type |
SRA |
| |
|
| Source name |
kidney
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: kidney
cell line: Human embryonic kidney 293 cells
treatment: lentiviral infection with shCtrl or shTHOC5, 1-3 days, then doxycycline for 12-16h and α-amanitin for 42h
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| Treatment protocol |
Infection of HEK293 cells with lentiviruses carrying either shCtrl or shTHOC5. The treatment was applied for 4 days. Transfection with siCtrl, siTHOC2, siTHOC6 or siCDK12 for 3 days.
|
| Growth protocol |
HEK293 cells were grown in Dulbecco´s modified Eagle's medium supplemented with 10% (v/v) FCS. HEK293 cells expressing Dox inducible mutant RNA Pol II that contain additional mutation for amanitine resistant (Fond et al ) were maintained in DMEM supplemented with 10% FBS, 200 μg/mL hygromycin B, 6.5 μg/mL blasticidin, and penicillin/streptomycin. Experiments using cells expressing Pol II mutants were performed after induction with 2.0 μg/mL doxycycline for 12–16 h and treatment with 2.5 μg/mL α-amanitin for a further 42 h, at which time all cell lines were viable, and endogenous Pol II was inactive.
|
| Extracted molecule |
cytoplasmic RNA |
| Extraction protocol |
Cytoplasmic RNA of HEK293 cells were achieved by RLN (Cyto-Buffer) and the Reliaprep™ miRNA Cell and Tissue Miniprep System (Promega). mRNAs were isolated by the NEBNext® Poly(A) mRNA Magnetic Isolation Module (E7490) with use of 30 µg RNA.
RNA libraries were prepared for sequencing using standard Nanopore protocols for Direct RNA sequencing (SQK-RNA002).
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| |
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
MinION |
| |
|
| Data processing |
Nanopore: Basecalls performed using Guppy version 3.3.3+fa743a6
Nanopore: Mapping using minimap2
Nanopore: Trimming of long read sequencing data using NanoFilt version 2.5.0
ChIp-Seq: FASTQ files were generated by CASAVA (v1.8.2). Galaxy workflow for RNA-Seq (www.usegalaxy.org) was used for subsequent data analysis. Reads were mapped to the human reference genome (GRCh38) using Bowtie2 (Galaxy Version 2.3.4.1). Metagene analysis was performed using deepTools 2.0
TT-Seq: Raw reads were mapped to the human reference genome (GRCh38) using Bowtie2 (Galaxy Version 2.3.4.1). Read coverage of individual gene was normalized and visualized using Seqmonk. To measure the progression of Pol II molecules into the gene body we applied a pipeline described by Gregersen et al. (33) for calling RNA Pol II transcription wave peak positions and elongation rates from DRB/TT-seq time-series data using R. Metagene analysis was performed using deepTools 2.0.
Assembly: GRCh38
Supplementary files format and content: For the identification of 3’cleavage sites the tool NanoFilt (Filtering and trimming of long read sequencing data) was used to trim the mRNA sequences from 5’end to a uniform length so that we keep always the last 200 nucleotides (python get_read_ends.py --bases_from_end 200 reads.fastq.gz | gzip > last_200_bp.fastq.gz). Trimmed reads were aligned to the GRCh38 human genome reference using minimap2. The quantification of 3’ cleavage was perform with Seqmonk using the human polyA database as reference. Genes that contain more than two annotated polyadenylation sites (PAS) and more than 5 reads at distal PAS sites were selected. The ratios between proximal- and distal cleavage site were calculated.
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| |
|
| Submission date |
Jun 21, 2022 |
| Last update date |
Dec 16, 2022 |
| Contact name |
Mareike Polenkowski |
| E-mail(s) |
[email protected]
|
| Organization name |
Medical School Hannover
|
| Department |
Cell Biochemistry
|
| Lab |
Tamura-Niemann
|
| Street address |
Carl-Neuberg-Straße 1
|
| City |
Hanover |
| ZIP/Postal code |
30625 |
| Country |
Germany |
| |
|
| Platform ID |
GPL24106 |
| Series (1) |
| GSE173374 |
THOC5 complexes with DDX5, DDX17 and CDK12 to regulate R loop structures and transcription elongation rate |
|
| Relations |
| BioSample |
SAMN29224671 |
| SRA |
SRX15815349 |
