|
| Status |
Public on Mar 16, 2023 |
| Title |
Taf13 degron, plus auxin, nascent RNA replicate 1 |
| Sample type |
SRA |
| |
|
| Source name |
Taf13-degron (SHY1043)
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
cell line: Taf13-degron (SHY1043)
cell type: S. cerevisiae
genotype: Taf13-degron (SHY1043)
treatment: 30 min auxin (500uM), 5 min 4-thiouracil (5mM)
|
| Treatment protocol |
Yeast cultures were treated with 500uM auxin (or DMSO alone for negative controls) for 30 minutes to degrade coactivators. Yeast cultures were then treated with 5mM 4-thiouracil (4-TU) to label nascent RNAs. Samples for RNA-seq were pelleted, snap frozen and stored at -80degC until processing. Samples for DNA-seq were collected immediately prior to auxin/DMSO treatment.
|
| Growth protocol |
UAS-core plasmid libraries were transformed into yeast using the LiOAC method. Yeast cultures were grown for several days with occasional dilution in synthetic complete (SC) media -Ura at 30degC to select for positive transformants. On the day of treatment, yeast cultures were grown to mid log phase (~0.8).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated using a RiboPure RNA Purification Kit (Thermo Fisher). Kit buffers were supplemented with 10mM BME. Nascent RNAs were purified using MTS-biotin and streptavidin beads as described in Duffy et al. 2015.
Nascent RNA barcodes were reverse transcribed with a reporter-specific primer. PCR1 was performed with primers complementary to the RT product and containing overhanging regions for multiplexing PCR. qPCR was used to determine the number of amplification cycles for PCR2. PCR2 was perfomed using unique dual indexes.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
NextSeq 2000 |
| |
|
| Description |
Nascent RNA (targeted)
|
| Data processing |
Consensus sequence counts were determined by clustering read 1 RNA library sequences using VSEARCH with 99% similarity.
Assembly: NA
Supplementary files format and content: Table. Column 1 "size" = number of sequence reads in cluster. Column 2 = consensus cluster sequence.
|
| |
|
| Submission date |
Nov 03, 2022 |
| Last update date |
Mar 16, 2023 |
| Contact name |
Jeremy Aspin Schofield |
| E-mail(s) |
jschofie@fredhutch.org
|
| Organization name |
Fred Hutchinson Cancer Center
|
| Department |
Basic Sciences Division
|
| Street address |
1100 Fairview Ave N
|
| City |
Seattle |
| State/province |
Washington |
| ZIP/Postal code |
98109 |
| Country |
USA |
| |
|
| Platform ID |
GPL31112 |
| Series (2) |
| GSE217229 |
Large-scale reporter assay to assess expression and coactivator specificity of UAS-core promoter combinations (RNA) |
| GSE217230 |
Large-scale reporter assay to assess expression and coactivator specificity of UAS-core promoter combinations |
|
| Relations |
| BioSample |
SAMN31592568 |
| SRA |
SRX18158297 |
