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| Status |
Public on Jun 02, 2024 |
| Title |
Spt3/7-degron yeast, plus auxin treatment |
| Sample type |
SRA |
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| Source name |
Spt3/7-degron (SHY1176)
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| Organism |
Saccharomyces cerevisiae |
| Characteristics |
cell line: Spt3/7-degron (SHY1176)
treatment: 500uM 3-IAA (30min), 5mM 4-TU (10min)
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| Treatment protocol |
For timecourse studies, WT yeast cells (BY4705) at mid-log phase were treated with 5 mM 4-TU (5mM) for 5min, 10min, or 15min or left untreated (DMSO only). Immediately following 4-TU treatment, cells were placed on ice and 2 volumes of ice-cold PBS was added to the culture. Cells were pelleted at 4°C and washed with ice cold PBS + 0.01% BSA. Cells were again pelleted and resuspended in 1mL 1ml PBS + 0.01% BSA. Cells were fixed by continuous gentle vortexing while adding ice cold methanol dropwise to a final concentration of 80%. Cells were incubated on ice for 15min, at which point fixed cells were stored overnight at –20°C. Coactivator depletion studies were performed in Med15-degron (SHY1055) or Spt3/7-degron (SHY1176) strains. Prior to 4-TU labeling for 10min, cells were treated with either 500uM auxin (3-indoleacetic acid) in DMSO or DMSO alone as a negative control, and the remainder of the above protocol was performed.
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| Growth protocol |
Saturated overnight cultures of yeast cells were diluted in fresh YPD media at an initial OD of 0.2. Cells were then cultured at 30°C to mid-log phase (OD ~0.8).
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
Fixed cells were incubated on ice for 15min, then pelleted and washed twice with ice cold PBS + 0.01% BSA. Cells were resuspended in 400ul PBS + 0.01% BSA, and the cell suspension was added to 1mL of alkylation buffer, followed by a 45min incubation at 45°C with occasional inversion. Cells were pelleted and washed twice with PBS + 0.01% BSA + 10mM DTT, then washed once with 1M sorbitol + 0.01% BSA. Cells were then resuspended in 900ul spheroplasting buffer, and 100ul of 100mg/ml Zymolyase 100T was added. Cells were spheroplasted for ~30min at 30°C with occasional inversion. The spheroplasting mixture was filtered through a MACS SmartStrainer, and spheroplasts were pelleted then washed twice in 1M sorbitol + 0.01% BSA. Spheroplasts were counted immediately prior to GEM generation using a hemocytometer.
The Chromium Next GEM Single Cell 3ʹ Kit (10x Genomics, V3.1) protocol was performed according to manufacturer's instructions. Briefly, GEMs containing Gel Beads decorated with poly(dT) capture sequences (including Illumina TruSeq Read 1 sequence, 10x Cell barcode, and UMI) were prepared. GEMs were combined with prepared yeast spheroplasts, resulting in lysis of the spheroplasts in droplets and capture of PolyA-containing RNA on Gel Beads. Captured RNAs were reverse transcribed, GEMs were broken and pooled, and cDNA was amplified resulting in the installation of i5 and i7 sample indexes. Prepared paired-end libraries were sequenced on an Illumina NextSeq 2000 instrument using P3 reagents, with read configuration of 28nt for R1 to identify cell barcode and UMIs, and 125nt for R2 to sequence the captured mRNA.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic single cell |
| Library selection |
cDNA |
| Instrument model |
NextSeq 2000 |
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| Description |
S37_plus
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| Data processing |
The CellRanger count pipeline (https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/what-is-cell-ranger) was performed on Fastq cells using a custom 3'-end S. cerevisiae annotation (derived from Roy and Chanfreau, 2020, PMID: 31128237) to generate a barcode, feature and matrix files.
Assembly: SacCer3
Supplementary files format and content: Tab-separated values files and matrix files
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| Submission date |
Nov 15, 2023 |
| Last update date |
Jun 02, 2024 |
| Contact name |
Jeremy Aspin Schofield |
| E-mail(s) |
jschofie@fredhutch.org
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| Organization name |
Fred Hutchinson Cancer Center
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| Department |
Basic Sciences Division
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| Street address |
1100 Fairview Ave N
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| City |
Seattle |
| State/province |
Washington |
| ZIP/Postal code |
98109 |
| Country |
USA |
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| Platform ID |
GPL31112 |
| Series (1) |
| GSE247795 |
Transcriptional noise, gene activation, and roles of SAGA and Mediator Tail measured using nucleotide recoding single cell RNA-seq |
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| Relations |
| BioSample |
SAMN38262007 |
| SRA |
SRX22530771 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7900893_S37_P_barcodes.tsv.gz |
21.1 Kb |
(ftp)(http) |
TSV |
| GSM7900893_S37_P_features.tsv.gz |
32.6 Kb |
(ftp)(http) |
TSV |
| GSM7900893_S37_P_matrix.mtx.gz |
7.2 Mb |
(ftp)(http) |
MTX |
SRA Run Selector |
| Raw data are available in SRA |
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