|
| Status |
Public on Mar 16, 2023 |
| Title |
Spt7 degron, plasmid library replicate 4 |
| Sample type |
SRA |
| |
|
| Source name |
Spt7-degron (SHY1045)
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
cell line: Spt7-degron (SHY1045)
cell type: S. cerevisiae
genotype: Spt7-degron (SHY1045)
treatment: none
|
| 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 |
genomic DNA |
| Extraction protocol |
Plasmid libraries were isolated from yeast transformants using a modified Mini Prep (Qiagen) protocol. Briefly, sample pellets were resuspended in buffer P1, added to tubes containing ~100ul silica beads, and physically lysed using a bead beater. The lysate was transferred to a fresh tube and the remainder of the Mini Prep protocol was followed.
PCR1 was performed with primers complementary to the reporter plasmid library 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 |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
NextSeq 2000 |
| |
|
| Description |
Plasmid DNA (targeted)
|
| Data processing |
Read pairs were joined into amplicons.
Consensus sequence counts were determined by clustering all DNA library sequences using VSEARCH with 97% 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) |
| GSE217227 |
Large-scale reporter assay to assess expression and coactivator specificity of UAS-core promoter combinations (DNA) |
| GSE217230 |
Large-scale reporter assay to assess expression and coactivator specificity of UAS-core promoter combinations |
|
| Relations |
| BioSample |
SAMN31592446 |
| SRA |
SRX18158137 |
