|
| Status |
Public on May 15, 2024 |
| Title |
Wt, 4tU, Rep1, Paf1CD |
| Sample type |
SRA |
| |
|
| Source name |
MATa his4-912δ lys2-128δ leu2Δ1 trp1Δ63
|
| Organisms |
Schizosaccharomyces pombe; Saccharomyces cerevisiae |
| Characteristics |
cell line: KY1021/KP03
treatment: None
time (minutes): NA
|
| Treatment protocol |
Once S. cerevisiae OD600 = 1.0-1.2 was reached, 4tU was added to 30 OD600 of culture to a final concentration of 0.65 mg/mL for precisely 5 min at RT before quenching by pouring cultures into 1/2 volume of dry-ice-cold methanol. Cells were pelleted and snap frozen before sonication.
|
| Growth protocol |
S. cerevisiae and S. pombe were grown in YPD standard rich growth medium supplemented with 400 μM tryptophan at 30°C.
|
| Extracted molecule |
other |
| Extraction protocol |
4tU-labeled S.cerevisiae pellets were spiked-in with labeled S. pombe at a 14:1 OD600 ratio and total RNA extracted with a hot acid-phenol extraction. 4tU-RNA labled was biotinylated with MTSEA-XX biotin and pulled down using Dynabeads MyOne Streptavidin C1 beads. RNA was purified using the RNEasy kit and quantified by Qubit.
4tU-seq libraires were prepared using the TECAN Ovation SoLo RNA-seq library preparation kit (TECAN, Redwood City, CA; 0516–32) targeting S. cerevisiae rRNAs for depletion using AnyDeplete technology. 2-10 ng of RNA was used per library build and fragment distributions assesed by tapestation
|
| |
|
| Library strategy |
OTHER |
| Library source |
other |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Description |
4tU-labled RNA
|
| Data processing |
Alignments were then converted to bam and duplicates were marked and removed using the samtools suite
S. cerevisiae Ribosomal RNA loci were blacklisted for subsequent analysis using bedtools intersect
Reads mapping to S. pombe were separated from those mapping to S. cerevisiae by selecting chromosomes to retain with samtools view
S. pombe reads over coding features were counted with Rsubread featureCounts
DESeq2 estimateSizeFactors() function used to generate normalization factors.
Bam files were converted to strand-specific bigWig format using deepTools2 bamCoverage using DESeq2-derived spike-in size factors.
Assembly: S. cerevisiae: Ensembl R64-3-1/S.pombe: ASM294v2 Hybrid Genome
Supplementary files format and content: bigwig file of spike-in normalized reads mapping to forward strand
Supplementary files format and content: bigwig file of spike-in normalized reads mapping to reverse strand
Library strategy: 4tU-seq
|
| |
|
| Submission date |
Feb 08, 2024 |
| Last update date |
May 15, 2024 |
| Contact name |
Karen Arndt |
| E-mail(s) |
arndt@pitt.edu
|
| Organization name |
University of Pittsburgh
|
| Department |
Biological Sciences
|
| Lab |
A316 Langley Hall
|
| Street address |
4249 Fifth Ave
|
| City |
Pittsburgh |
| State/province |
PA |
| ZIP/Postal code |
15260 |
| Country |
USA |
| |
|
| Platform ID |
GPL28173 |
| Series (1) |
| GSE255360 |
Dynamic Roles for the Paf1 Complex in Regulating Transcription Elongation and Co-Transcriptional Processes [4tU-seq] |
|
| Relations |
| BioSample |
SAMN39888901 |
| SRA |
SRX23576113 |
