 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Apr 26, 2024 |
| Title |
PolySeq~tg_17~L114~PolySeq_0.8%Azide~BY4741~30C~30min~mock~BR1~Poly |
| Sample type |
SRA |
| |
|
| Source name |
yeast cells
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
cell type: yeast cells
|
| Treatment protocol |
NA
|
| Growth protocol |
Cells were grown overnight at 30°C with shaking for at least 12 hours to OD600 = 0.4 in synthetic complete dextrose media (SCD) before being exposed to stress.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Around 100 mg of frozen yeast that was collected by vacuum filtration was transferred to a pre-chilled 2 ml Eppendorf Safe-Lock tube. Cells were lysed with a pre-chilled 7 mM stainless steel ball (Retsch #05.368.0035) by 5x90sx30Hz pulses in a Retsch MM100 mixer mill, chilling in liquid nitrogen (LN2) between pulses. Sample was resuspended in 10:1 (v/w) polysome lysis buffer (20 mM HEPES-KOH (pH 7.4), 100 mM KCl, 5 mM MgCl2, 200 μg/mL heparin (Sigma #H3149), 1% triton X-100, 0.5 mM TCEP (Goldbio #TCEP25), 100 μg/mL cycloheximide (Sigma #C7698-5G), 20 U/ml SUPERase•In (Invitrogen #AM2696), 1:200 Millipore protease inhibitor IV #539136). The lysate was clarified by centrifugation at 3000 g for 30 s, and the clarified lysate was transferred to new tube and aliquots were flash frozen in LN2. A 10–50% continuous sucrose gradient in polysome gradient buffer (5 mM HEPES-KOH (pH 7.4), 140 mM KCl, 5 mM MgCl2, 100 μg/ml cycloheximide, 10 U/ml SUPERase•In, 0.5 mM TCEP) was prepared in SW 28.1 tubes (Seton #7042) using a Biocomp Gradient Master and allowed to cool to 4°C. Clarified lysate (200 µL) was loaded on top of the gradient, and gradients were spun in a SW28.1 rotor at 27,500 rpm for 3.5 hr at 4°C. Gradients were fractionated into 0.6mL fractions using a Biocomp Piston Gradient Fractionator with UV monitoring at 254 nm, and fractions were flash frozen in LN2. UV traces were normalized to the total signal starting with the 40S peak. The samples were generated by pooling 50 µL of each fraction from the free fraction (before the monosome peak) and either separately pooling the fractions with 3+ ribosomes bound and the mono/di-some fractions (for the heat shock experiments), or by combining all ribosome-bound fractions together (azide and ethanol stresses). The spike-in (50 ng of S. pombe total RNA) was then added to each pooled sample. RNA was purified via ethanol precipitation (final concentrations of 0.3 M sodium acetate pH 5.2, 0.3 µg/mL glycoblue (Invitrogen #AM9516), and 70% ethanol) at -20°C overnight followed by centrifugation at 4°C for 30 minutes at 21,000 g. The pellet was washed with 1 mL of 70% ethanol before being resuspended in water. The purified RNA was then treated with Dnase I (NEB) before purifying again using an NEB RNA clean and concentrate kit (NEB #T2030).
Sequencing libraries were prepared by the University of Chicago Genomics Facility from DNase I treated RNA using Qiagen FastSelect (Qiagen #334215) and Illumina Stranded mRNA Prep (Illumina #20020595) kits. Paired end 200 bp sequencing was performed on an Illumina NovaSeq 6000.
In general, cells were first lysed by cryo-grinding with a steel ball. When appropriate, fractionation by sedimentation or sucrose gradients was performed and RNA was dissolved in Trizol. The RNA was then purified using Zymo Direct-Zol columns. For more details, please see the protocol associated with each sample.
In general, RNA sequencing libraries were prepared using either Illumina RNA sequencing kits or NEBNext Ultra II RNA sequencing kits.
|
| |
|
| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Data processing |
Sequencing reads were trimmed using TrimGalore (v0.6.10, https://github.com/FelixKrueger/TrimGalore) using default settings (e.g. trim_galore --paired --fastqc_args '--outdir fastqc/' -j 4 -o trimmed --basename F02 AD-JB-F02_S44_R1_001.fastq.gz AD-JB-F02_S44_R2_001.fastq.gz). They were mapped using STAR v2.7.10b(Dobin et al. 2013) (e.g. STAR --outSAMtype BAM Unsorted --readFilesCommand gunzip -c --sjdbGTFfile spike_saccharomyces_cerevisiae_R64-3-1_20210421_geneid.gff3 --sjdbGTFtagExonParentTranscript Parent --sjdbGTFfeatureExon CDS --sjdbGTFtagExonParentGene gene_id --runThreadN 4 --alignMatesGapMax 20000 --limitBAMsortRAM 1445804817 --genomeDir STAR_spike_saccharomyces_cerevisiae_R64-3-1_20210421 --outFileNamePrefix mapped_reads/F02/F02_ --readFilesIn trimmed/F02_val_1.fq.gz trimmed/F02_val_2.fq.gz). The estimated counts and TPMs were generated using kallisto v0.48.0(Bray et al. 2016) (e.g. kallisto quant -i spike_Scerevisiae_orf_coding_all_Scerevisiae_rna_coding.fasta.idx -o kallisto_quant/F02 --fr-stranded --bootstrap-samples=50 -t 1 trimmed/F02_val_1.fq.gz trimmed/F02_val_2.fq.gz).
S. pombe total RNA was added and reads were mapped to saccharomyces_cerevisiae_R64-3-1_20210421_Schizosaccharomyces_pombe_all_chromosomes_relabelled.fasta (STAR) using the annotation file saccharomyces_cerevisiae_R64-3-1_20210421_Schizosaccharomyces_pombe_all_chromosomes_relabelled_geneid.gff3, and Scerevisiae_orf_coding_all_Scerevisiae_rna_coding_Spombe_cds.fasta (kallisto).
In general, sequencing reads were mapped to the transcriptome using kallisto to extract estimated counts and TPMs.
Assembly: S288C R64.3.1
Supplementary files format and content: estimated counts and TPMs per RNA sample
|
| |
|
| Submission date |
Apr 26, 2024 |
| Last update date |
Apr 26, 2024 |
| Contact name |
D Allan Drummond |
| E-mail(s) |
dadrummond@uchicago.edu
|
| Organization name |
University of Chicago
|
| Street address |
929 E 57th Street
|
| City |
Chicago |
| State/province |
IL |
| ZIP/Postal code |
60637 |
| Country |
USA |
| |
|
| Platform ID |
GPL27812 |
| Series (1) |
| GSE265963 |
Transcriptome-wide mRNA condensation precedes stress granule formation and excludes stress-induced transcripts |
|
| Relations |
| BioSample |
SAMN41096666 |
| SRA |
SRX24381089 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM8238320_JB194_abundance.tsv.gz |
206.3 Kb |
(ftp)(http) |
TSV |
SRA Run Selector |
| Raw data are available in SRA |
|
|
|
|
 |
