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| Status |
Public on Feb 12, 2024 |
| Title |
Nab3-AID, quiescence, day7, nascent RNA, 7718, replicate 2 |
| Sample type |
SRA |
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| Source name |
W303
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| Organism |
Saccharomyces cerevisiae |
| Characteristics |
cell line: W303
genotype: Nab3-AID
treatment: IAA powder (98% purity, Sigma-Aldrich) was added at a concentration of 1 mg/mL.
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| Growth protocol |
Quiescent cells were grown for 7 days to saturation in YEP with 2% glucose and then purified using a gradient method.
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| Extracted molecule |
total RNA |
| Extraction protocol |
To prepare 4tU-seq libraries, we employed the Universal Plus™ Total RNA-Seq library preparation kit and custom yeast rRNA depletion (AnyDeplete®).
RNA was extracted using yeast RiboPure™ RNA Purification Kit from Invitrogen™
4tU-seq
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| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
NextSeq 2000 |
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| Description |
R1 and R3 are, respectively, forward and reverse reads; R2 are UMI indices.
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| Data processing |
Paired-end reads underwent quality assessment with FastQC and MultiQC. Unique Molecular Identifier (UMI) sequences were appended to the reads using UMI-tools, and adapter and quality trimming were performed with Atria. For nascent transcriptome assembly, reads were additionally subjected to k-mer adjustment using Rcorrector. For more details, see Greenlaw et al.
Using the aligner STAR, processed reads were aligned to a concatenated multi-organism reference genome consisting of the S. cerevisiae, K. lactis, and 20S narnavirus genomes. Non-k-mer-adjusted bams (*.UTPD.bam) were indexed and filtered (with Samtools) to retain only primary alignments, and technical duplicates were removed using UMI-tools. k-mer-adjusted bams (*.UTKPSSc.bam) were indexed and filtered to retain both primary and secondary alignments; alignments associated with the K. lactis and 20S narnavirus genomes were then filtered out. For more details, see Greenlaw et al.
Gene Transfer Format (gtf) and Gene Feature Format (gff3) files were obtained or derived from publicly available sources using custom scripts. Additionally, some files were generated via nascent transcriptome assembly and annotation using Trinity, GffRead, and custom scripts. To perform feature-level quantification, we used htseq-count with the gtf and gff3 files in conjunction with processed non-k-mer-adjusted bams. For more details, see Greenlaw et al.
To generate normalized coverage tracks (*.bw), we used processed non-k-mer-adjusted bams as input to the utility bamCoverage (deepTools) in one of two ways: (a) For the bins per million (BPM) normalization method, mapped reads were normalized by their library size while excluding all rRNA and tRNA regions. The bin size for this method was set to 1 base pair. (b) For the K. lactis scaled coverage (SC) method, library size estimates were first calculated using K. lactis spike-in counts. The bam files were then processed by supplying the reciprocals of the estimates as arguments (--scaleFactor) to bamCoverage. Again, the bin size was set to 1 base pair. For more details, see Greenlaw et al.
Assembly: sacCer3 R64-1-1
Supplementary files format and content: bams for individual replicates (non-k-mer-adjusted and k-mer-adjusted) and merged replicates (k-mer-adjusted)
Supplementary files format and content: bw files for individual and merged replicates, derived from non-k-mer-adjusted bams, and normalized using either the BPM calculation or the K. lactis spike-in-scaled coverage calculation
Supplementary files format and content: gtf and gff3 files obtained or derived from publicly available sources, or generated via custom transcriptome assembly and annotation
Supplementary files format and content: tab-delimited text files containing sample-specific raw counts for features in the various gtf and gff3 files
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| Submission date |
Jul 28, 2023 |
| Last update date |
Feb 12, 2024 |
| Contact name |
Alison C Greenlaw |
| E-mail(s) |
alisoncg37@gmail.com
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| Phone |
4259859567
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| Organization name |
Fred Hutch Cancer Center
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| Department |
Basic Sciences
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| Lab |
Tsukiyama
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| Street address |
1100 Fairview Ave N
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| City |
Seattle |
| State/province |
WA |
| ZIP/Postal code |
98109 |
| Country |
USA |
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| Platform ID |
GPL31112 |
| Series (1) |
| GSE239568 |
Post-transcriptional regulation shapes the transcriptome of quiescent yeast |
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| Relations |
| BioSample |
SAMN36748613 |
| SRA |
SRX21185130 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7667343_Nab3-AID_Q_day7_tcn_N_auxT_tcF_7718_rep2_batch1.KLSC_m.bw |
45.6 Mb |
(ftp)(http) |
BW |
| GSM7667343_Nab3-AID_Q_day7_tcn_N_auxT_tcF_7718_rep2_batch1.KLSC_p.bw |
45.3 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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