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| Status |
Public on Apr 26, 2024 |
| Title |
SedSeq~tg_4~L58~eIF4E_deplete~yHG038~42C~10min~mock~BR1~Total |
| Sample type |
SRA |
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| Source name |
yeast cells
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| Organism |
Saccharomyces cerevisiae |
| Characteristics |
cell type: yeast cells
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| Treatment protocol |
For depletion experiments, yeast were grown at 30°C in YPD to OD600 = 0.1. To induce TIR1 ligase, 5 µM β-estradiol (10 mM stock in DMSO) or an equivalent volume of DMSO (for mock treatment) was added to each culture and they were incubated for 75 minutes. To induce degradation, either 100 µM of Indole-3-acetic acid sodium salt (Sigma #I5148, 250 mM stock in DMSO) or 5 µM of 5-Ph-IAA (Medchemexpress #HY-134653, 5 mM stock in DMSO) was added. After 2 hours of auxin exposure, the culture was centrifuged and the yeast pellet was incubated in either 42°C or 46°C water baths for 10 minutes. Control cells were placed inside a 30°C incubator.
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| 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.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Biochemical fractionation was completed similarly to Wallace et al. [2015], with the major exception that 20,000 g for 10 min was used rather than the original 100,000 g for 20 min. In short, 50 mL cultures of treated yeast were harvested by centrifugation at 3000 g for 5 minutes, then resuspended in 100 µL of soluble protein buffer (SPB: 20 mM HEPES, pH 7.4, 140 mM KCl, 2 mM EDTA, 0.1 mM TCEP, 1:200 protease inhibitor (Millipore #539136), 1:1000 SUPERase•In RNase Inhibitor (Invitrogen #AM2696)), and flash frozen in liquid nitrogen as a pellet in a 2 mL Eppendorf Safe-Lock tube (Eppendorf #0030123620) with a 7 mm steel ball (Retsch #05.368.0035). The cells were then lysed using a Retsch MM400 for 5x90s at 30 Hz, chilling in liquid nitrogen between each shaking repeat. The lysed cells were resuspended in 600 µL of SPB, and 100 µL of total sample was transferred to 300 µL of Trizol LS (Invitrogen #10296010). The remainder was centrifuged for 30 seconds at 3000 g, and 300 µL of clarified lysate was transferred to a new 1.5 mL tube. This was then centrifuged for 10 minutes at 20,000 g. A 100 µL supernatant sample was transferred to 300 µL of Trizol LS, and 400 µL of SPB was added to the pellet as a wash. After another spin at 20,000 g for 10 minutes, the supernatant was removed and the pellet was resuspended by vortexing for 15 minutes in 300 µL of Trizol LS and 100 µL of water. If required, 1 ng of spike-in nanoluc transcript was added to each sample at this step before RNA was isolated using Zymo Direct-Zol RNA extraction columns (Zymo #R2052), and RNA integrity was assessed by the appearance of two sharp rRNA bands on a 1% agarose gel and quantified using the absorbance at 260 nm.
Sequencing libraries were prepared by from DNase I treated RNA using Qiagen FastSelect (Qiagen #334215), NEBNext Multiplex Oligos (UMI Adaptor RNA Set 1, NEB #E7335L) and NEBnext Ultra II Directional RNA library prep kits (NEB #E7760L). Paired end 200 bp sequencing with additional reads for dual 8/8 indices plus the 11nt UMI after the i7 index was performed on an Illumina NovaSeq 6000 at the University of Chicago Genomics Facility.
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.
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| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina NovaSeq 6000 |
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| Data processing |
The unique molecular indices (UMIs) were extracted from fastq R2 using Umi-Tools v1.1.4(Smith et al. 2017) and stored in fastq R1 and R3 (e.g. umi_tools extract --bc-pattern=XXXXXXXXNNNNNNNNNNN -I AD-JB-1S-HG02_S2_R2_001.fastq.gz --read2-in=AD-JB-1S-HG02_S2_R1_001.fastq.gz --read2-out=labeled_fastq/HG002/HG002_R1.umi.fastq. Sequencing reads were then trimmed using TrimGalore (v0.6.10, https://github.com/FelixKrueger/TrimGalore) using default settings (e.g. trim_galore --paired --gzip --fastqc_args '--outdir fastqc/' -j 4 -o trimmed --basename HG002 labeled_fastq/HG002/HG002_R1.umi.fastq labeled_fastq/HG002/HG002_R3.umi.fastq). 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/HG002/HG002_ --readFilesIn trimmed/HG002_val_1.fq.gz trimmed/HG002_val_2.fq.gz). Umi-Tools was then used again to deduplicate the reads (e.g. umi_tools dedup --stdin=mapped_reads/HG002/HG002_Aligned_Sorted.out.bam --chimeric-pairs=discard --unpaired-reads=discard --spliced-is-unique --paired -S mapped_reads/HG002/HG002_Aligned.sortedByCoord.dedup.out.bam). The reads were split again into fastq files using samtools v1.16.1(Li et al. 2009), and then 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/HG002 --rf-stranded --bootstrap-samples=50 -t 1 mapped_reads/HG002/HG002_Aligned_dedup_R1.fastq.gz mapped_reads/HG002/HG002_Aligned_dedup_R3.fastq.gz).
NanoLuc spike-in RNA was added and reads were mapped to spike_Scerevisiae_orf_coding_all_Scerevisiae_rna_coding.fasta (STAR) using the annotation file spike_saccharomyces_cerevisiae_R64-3-1_20210421_geneid.gff3, and spike_Scerevisiae_orf_coding_all_Scerevisiae_rna_coding.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
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| Submission date |
Apr 26, 2024 |
| Last update date |
Apr 26, 2024 |
| Contact name |
D Allan Drummond |
| E-mail(s) |
dadrummond@uchicago.edu
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| Organization name |
University of Chicago
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| Street address |
929 E 57th Street
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| City |
Chicago |
| State/province |
IL |
| ZIP/Postal code |
60637 |
| Country |
USA |
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| Platform ID |
GPL27812 |
| Series (1) |
| GSE265963 |
Transcriptome-wide mRNA condensation precedes stress granule formation and excludes stress-induced transcripts |
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| Relations |
| BioSample |
SAMN41096832 |
| SRA |
SRX24381037 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM8238154_HG137_abundance.tsv.gz |
85.6 Kb |
(ftp)(http) |
TSV |
SRA Run Selector |
| Raw data are available in SRA |
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