|
| Status |
Public on Feb 09, 2021 |
| Title |
tNetSeq R749H slow pol II Rep1 |
| Sample type |
SRA |
| |
|
| Source name |
HEK293 Flp-in T-REx pcDNA5 Rpb1 Amr R749H
|
| Organism |
Homo sapiens |
| Characteristics |
growth conditions: DMEM 10% FCS, 200μg/ml hygromycin B, 6.5μg/ml blasticidin, pen/strep.
antibody: rabbit anti pan CTD (Schroeder eta l Genes Dev. 14:2435, 2000)
genotype: R749H slow pol II
treatment: untreated
cell line: HEK293
|
| Treatment protocol |
Amr Rpb1 was induced with doxycycline (2.0 ug/ml) for 12-16 hr then treated with α-amanitin (2.5 mg/ml) for a further 42 hr before harvesting.
|
| Growth protocol |
Rpb1 WT and mutant cells were grown in DMEM 10% FCS, 200μg/ml hygromycin B, 6.5μg/ml blasticidin, pen/strep.
|
| Extracted molecule |
nuclear RNA |
| Extraction protocol |
[tNet_MaPseq] IP's were prepared from DNAseI solublilized nuclear extracts (~1.5X 10E7 cells). Precipitated RNA was was purified by Trizol extraction and random primed. DMS and DMSO treated RNA was reverse transcribed under MaP conditions. All tNetSeq and tNetMaP-Seq libraries were constructed using the KAPA stranded RNAseq kit.
[tNet_PIPseq] Following RNAase digestion, samples with treated with Proteinase K at 1mg/ml for 15mins at 37º and then 65 º for 30mins to reverse crosslinks. RNA was purifed with trizol extraction and RNA fragments were convereted into sequencing libraries with lexogen small RNA kit.
|
| |
|
| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Description |
nascent nuclear RNA
Anti-pol II nascent RNA
|
| Data processing |
Library strategy: tNET-Seq
Paired-end 150 base reads (after removing barcodes and trimming adaptor sequences) were uniquely mapped to the hg19 UCSC human genome hisat2
Genome_build: GRCh37/hg19 assembly
Supplementary_files_format_and_content: Data sets were processed into Bedgraph files. tNet-MaP seq, tNetPIP seq and tNET-seq edits were processed using pipelines available at https://github.com/rnabioco/rnastruct. Putative protein footprints were subtracted in files labelled no FP. Structure score files that include reads mapped to repetitive elements are labelled non-unique. DMS files were background subtracted (bgsub).
|
| |
|
| Submission date |
Apr 21, 2020 |
| Last update date |
Feb 09, 2021 |
| Contact name |
David Bentley |
| E-mail(s) |
David.Bentley@ucdenver.edu
|
| Phone |
3037243237
|
| Organization name |
UCD AMC
|
| Department |
BMG
|
| Lab |
Bentley
|
| Street address |
12801 E. 17th Ave, RC1 So. RM 10401D
|
| City |
Aurora |
| State/province |
Colorado |
| ZIP/Postal code |
80045 |
| Country |
USA |
| |
|
| Platform ID |
GPL24676 |
| Series (1) |
| GSE149018 |
Alternative RNA Stuctures Formed During Transcription Depend on Elongation Rate and Modify RNA processing |
|
| Relations |
| BioSample |
SAMN14652327 |
| SRA |
SRX8148245 |
