|
| Status |
Public on Jan 08, 2020 |
| Title |
WT_Rpb3Flag_DMSO_B |
| Sample type |
SRA |
| |
|
| Source name |
whole organism
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
strain: SHY1236
genotype: mat alpha delta ade2::hisG his3 delta 200 leu2 delta 0 lys2 delta 0 met15 delta 0 trp1 delta 63 ura3 delta 0 RPB3-3x Flag::NAT MX pGPD1-OSTIR::HIS3
treatment: DMSO for 30 minutes followed by 6 minute RNA labeling with 4-tU
growth: 30 degrees in synthetic medium to mid-log phase
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Newly synthesized RNAs were labeled as previously described (Bonnet et al., 2014; PMID: 25228644). S. cerevisiae and previously labeled S. pombe cells were mixed in an 8:1 ratio and total RNA was extracted using the RiboPure yeast kit (Ambion, Life Technologies) using the following volumes: 480 μl lysis buffer, 48 μl 10% SDS, 480 μl phenol:CHCl3:IAA (25:24:1) per S. cerevisiae pellet + 50 μl S. pombe (from a single S. pombe pellet resuspended in 850 μl lysis buffer). Total RNA was then biotinylated and purified essentially as described (Duffy et al., 2015; PMID: 26340425; Duffy and Simon, 2016; PMID: 27925666) using 40 μl (~40 μg) total RNA. In the last step 50 μl each of RNA sample eluted from streptavidin beads was adjusted to 100 μl with nuclease-free water and purified on RNeasy columns (Qiagen) using the modified protocol as described (Duffy and Simon, 2016; PMID: 27925666). RNAs were eluted into 14 μl nuclease-free water.
Ovation Universal RNA-seq System kits (Tecan)
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Data processing |
Paired-end sequencing reads were aligned to S. cerevisiae reference genome (sacCer3) and S. pombe reference genome (ASM294v2.20) with Bowtie2 using optional arguments “-I 10 -X 700 --local --very-sensitive-local --no-unal --no-mixed --no-discordant”.
SAM files for S. cerevisiae data were used as an input for HTseq-count with default settings. The GFF file with S. cerevisiae genomic features was downloaded from the Ensembl website.
Signal per gene was normalized by the number of all S. pombe reads mapped for the sample and multiplied by 10000 (arbitrarily chosen number). Genes classified as dubious, pseudogenes or transposable elements were excluded leaving 5794 genes for the downstream analysis.
Genome_build: sacCer3 (S. cerevisiae), ASM294v2.20 (S. pombe)
Supplementary_files_format_and_content: Processed CSV files contain normalized signal for all analyzed features (5794 mRNA genes).
|
| |
|
| Submission date |
Dec 17, 2019 |
| Last update date |
Jan 08, 2020 |
| Contact name |
Rafal Donczew |
| E-mail(s) |
Rafal-donczew@omrf.org
|
| Organization name |
Oklahoma Medical Research Foundation
|
| Department |
Cell Cycle and Cancer Biology
|
| Lab |
Hahn Lab
|
| Street address |
825 NE 13th St
|
| City |
Oklahoma City |
| State/province |
OK |
| ZIP/Postal code |
73104 |
| Country |
USA |
| |
|
| Platform ID |
GPL17342 |
| Series (2) |
| GSE142122 |
Two separate roles for the transcription coactivator SAGA and a set of genes redundantly regulated by TFIID and SAGA |
| GSE142182 |
Two separate roles for the transcription coactivator SAGA and a set of genes redundantly regulated by TFIID and SAGA [RNA-Seq] |
|
| Relations |
| BioSample |
SAMN13614521 |
| SRA |
SRX7397025 |
