|
| Status |
Public on Jan 21, 2022 |
| Title |
Log Growth Replicate 3 |
| Sample type |
SRA |
| |
|
| Source name |
S. cerevisiae
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
strain: pJD47
time point: LOG
|
| Treatment protocol |
Proliferating cultures were pelleted and resuspended in autoclaved water containing 10 mM D3-acetate (Sigma 176079) to induce quiescence. After 7 days, quiescent cultures were pelleted and washed to remove any residual D3-acetate, and proliferation was induced by resuspending in liquid media.
|
| Growth protocol |
Saccharomyces cerevisiae glycerol stocks were streaked onto 2% agar plates containing yeast nitrogen base (Difco #233520), synthetic complete amino acids mix (Bufferad #S0051) and 2% glucose. Following overnight incubation at 30°C, starter cultures were inoculated with single colonies and incubated overnight at 30°C. Secondary cultures were inoculated at OD600=0.2 and grown at 30°C until they reached OD600=1.
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
Cultures were pelleted and resuspended in QIAzol (Qiagen). Cells were lysed using a 0.5 mm Zirconia/Silica beads (RPI #9834) in a Mini-Beadbeater high energy cell disruptor (BioSpec) at 4°C, for 4 times 60 seconds. Samples were cooled on ice for 2 min in-between beatings. RNA was subsequently purified using RNeasy Mini kit (Qiagen).
1 ug of RNA was used to construct sequencing libraries using the NEBNext Ultra II RNA library preparation kit for Illumina (New England Biolabs; NEB). Libraries were multiplexed using NEBNext Multiplex Oligos for Illumina (dual index primers) and single-end sequenced (75 bp) on the NextSeq 550 platform (Illumina) in accordance with the manufacturer’s protocol.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
NextSeq 550 |
| |
|
| Data processing |
Demultiplexing: sequencing data were demultiplexed using native applications on BaseSpace.
Alignment: demultiplexed FASTQs were aligned by RNA-STAR v.2.5.2a to the sacCer3 assembly of the yeast genome (parameters --outFilterType BySJout --outFilterMultimapNmax 20 --alignSJoverhangMin 8 --alignSJDBoverhangMin 1 --outFilterMismatchNmax 999 --alignIntronMin 20 --alignIntronMax 1000000).
Gene Quantification: aligned reads were mapped to genomic features using HTSeq v.0.6.1 after merging four lanes of NextSeq (parameters -r pos -s no -t exon -i gene_id).
Size Adjustment: library size adjustment and analysis of differential gene expression was done using DESeq2 (Wald’s test) using the time-course variable in the design; adjusted counts supplied in the Adjusted Values.txt table were obtained using counts(DESEQ_OBJECT, normalized=T)
Track Creation: supplied bigWigs were obtained by first pooling three replicates in each time point, then employing the BEDtools script genomeCoverageBed -bg to create a coverage map (bedGraph) of pooled RNA-seq signal, then multiplying tag counts in the map by the RPM coefficient for each pooled time point. Size-adjusted bedGraphs were then sorted into position order and rendered as bigWigs using the UCSC Genome Browser utilities bedSort and bedGraphToBigWig.
Genome_build: sacCer3
Supplementary_files_format_and_content: The supplied bigWig files are tracks showing the RPM-adjusted RNA-seq tag counts over the genome; the Adjusted Values.txt file contains tag counts per-gene, per-sample adjusted for library size using DESeq2.
|
| |
|
| Submission date |
Nov 23, 2021 |
| Last update date |
Jan 21, 2022 |
| Contact name |
Gregory Donahue |
| Organization name |
The University of Pennsylvania
|
| Department |
Cell & Developmental Biology
|
| Lab |
Zaret Lab
|
| Street address |
3400 Civic Center Blvd, Bldg 421
|
| City |
Philadelphia |
| State/province |
PA |
| ZIP/Postal code |
19104 |
| Country |
USA |
| |
|
| Platform ID |
GPL26302 |
| Series (2) |
| GSE189444 |
Enzymatic transfer of acetate on histones from lysine reservoir sites to lysine activating sites (RNA-Seq) |
| GSE189446 |
Enzymatic transfer of acetate on histones from lysine reservoir sites to lysine activating sites |
|
| Relations |
| BioSample |
SAMN23413061 |
| SRA |
SRX13211218 |
