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Status |
Public on Dec 24, 2020 |
Title |
KL_AS_M24_cont_4-2 |
Sample type |
SRA |
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Source name |
exponential growth in YPD
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Organism |
Kluyveromyces lactis |
Characteristics |
analog_sensitive: TRUE deleted_tf: MSN2_MSN4 treatment: DMSO
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Treatment protocol |
The culture was divided into two 12ml treatment and control cultures and 3.6µl of 10mM 1-NM-PP1 (for a final concentration of 3µM) or DMSO was added respectively. Each culture was incubated while shaking at 250RPM and 30°C for 50 min prior to collection.
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Growth protocol |
Cells were grown overnight at 30°C in YPD to saturation, and then diluted to obtain a 30ml of cells with an OD600 of 0.5 in 4-6 hours assuming a lag time of 90 min and a doubling time of 90 min for S. cerevisiae and 110min for K. lactis.
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Extracted molecule |
polyA RNA |
Extraction protocol |
Cultures were split into two 5ml aliquots prior to spinning down at 3850RPM (331rcf) on an Eppendorf 5810R benchtop centerfuge for 3min. After spinning down, supernatants were poured out and remaining supernatant was aspirated off with a P1000 pipette. Cells were then flash frozen in liquid nitrogen and stored at -80°C. RNA extraction was performed using the hot acid-phenol extraction protocol of (Solís et al. 2016) with the following changes. Initial cell volume was 5ml instead of 1.5ml and thus the initial spin for collecting the cells was done for 3min at 3850RPM (331rcf) instead of 30s at 13000RPM (15871rcf). Acid-phenol:chloroform:isoamyl alcohol (IAA) (125:24:1), pH 4.5 (AM9722) was used instead of pure Acid-Phenol because the chloroform aids in the separation of nucleic acid from proteins and lipids, and the IAA prevents foaming. The spin following heat incubation was performed at room temperature instead of at 4°C. A second 400µl chloroform wash was included prior to removing the aqueous phase from the phase lock tubes. 22µl of 3M NaOAc, pH5.2 was added instead of 30µl to precipitate the DNA. Ethanol Precipitation was done overnight instead of for 30 min. 3’ Sequencing Libraries were prepared using the Lexogen QuantSeq 3’mRNA-Seq Library Prep kit FWD using dual indices for each sample. Select libraries were checked for quality on the Bioanalyzer using a High Sensitivity DNA chip. Library concentrations were calculated using a Qbit 2.0 Fluorometer (Invitrogen), and 2.65 ng per sample were pooled and sequenced on an Illumina Hiseq 4000 sequencer to an average depth of between 245,000 and 5.6 million reads per sample (median 2.53 million).
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Library strategy |
RNA-Seq |
Library source |
transcriptomic |
Library selection |
cDNA |
Instrument model |
Illumina HiSeq 4000 |
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Data processing |
Sequencing data was trimmed, aligned, and checked for quality using the Bluebee genomics analysis pipeline. S. cerevisiae samples were run using the “FWD S. cerevisiae (R64) Lexogen QuantSeq 2.2.3” protocol, and K. lactis samples were run using the “FWD K. lactis (ASM241v1) Lexogen Quantseq2.2.3” protocol Counts were generated from the resultant fastq files using custom GTFs that included 3'UTR annotations and the CDS of each gene. For S. cerevisiae 3'UTRs were identified using information from Nagalakshmi et al. 2008, and for K. lactis the 3'UTR was estimated to be 400bp downstream of the stop codon for the purpose of assigning counts to genes. Counts were then generated from the .bam files using htseq in intersection-nonempty mode. See https://github.com/heineike02/UTR_annotation for details on the custom GTF files. Gene count data was processed to yield estimates for raw expression (rlog values) for all experiments as well as Log Fold Change and adjusted pValue estimates for differential expression between conditions using the DESeq2 package in R (Love, Huber, and Anders 2014). After filtering samples that failed quality control, our RNA-seq dataset contained 59 S. cerevisiae samples and 35 K. lactis samples which were sequenced in the same run. These samples included the WT +/- drug and AS +/- drug experiments as well as experiments with Msn2/4 deletion and Rph1/Gis1 deletion mutants. In each species, we kept only genes that had 2 or more counts in at least 3 samples for that species. The rlog was calculated using the rlog function with blind=FALSE, and an experimental design which combined the presence of the AS mutation, transcription factor deletion status and presence of drug into a single factor. Mean rlog data for each condition was calculated by averaging rlog values across replicates. To calculate distributions of rlog values for all genes in S. cerevisiae, 443 orfs classified as dubious in the saccharomyces_cerevisiae_R64-2-2_20170117 GFF were filtered out. Log Fold Change and adjusted p-values for differential expression between samples was calculated from deseq using a False Discovery Rate of 1% for the relevant contrast (PKA-AS strains +/- drug at 50 min, or ΔMsn2/4 or ΔKL.Msn2 PKA-AS strains +/- drug at 50 min). Custom scripts for further RNA sequencing analysis and enrichment are located at https://github.com/heineike02/yeast_esr_expression_analysis Genome_build: S. cerevisiae: R64, K. lactis: ASM241v1 stored in Bluebee genomics protocols 26 Oct 2018. Supplementary_files_format_and_content: read_count .txt files are tab separated and contain gene names and counts for each gene. Matrices containing count data for each species are also included (20181017_countdata_postQC_KL.csv and 20181017_countdata_postQC_SC.csv). Supplementary_files_format_and_content: Tables containing LFC and pvalues for AS strains contrasting 1-NM-PP1 vs control treatment as generated by DESEQ2 are also included for each species either with no transcription factor mutation (20200603_deseq_KL_AS_WT_nmpp1.csv and 20200603_deseq_SC_AS_WT_nmpp1.csv) or with the Msn2/4 deletion (20201003_deseq_KL_AS_M24_nmpp1.csv and 20201003_deseq_SC_AS_M24_nmpp1.csv). Supplementary_files_format_and_content: Tables containing mean rlog values as generated by DESEQ2 for all conditions are included (20200603_rlog_mean_KL.csv and 20200603_rlog_mean_SC.csv)
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Submission date |
Dec 23, 2020 |
Last update date |
Dec 24, 2020 |
Contact name |
Hana El-Samad |
E-mail(s) |
hana.el-samad@ucsf.edu
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Organization name |
University of California San Franicisco
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Department |
Biochemistry and Biophysics
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Lab |
El-Samad Lab
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Street address |
1700 4th Street, Box 2542
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City |
San Francisco |
State/province |
CA |
ZIP/Postal code |
94158-2330 |
Country |
USA |
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Platform ID |
GPL22716 |
Series (1) |
GSE163741 |
Protein Kinase A (PKA) inhibition in Saccharomyces cerevisiae and Kluyveromyces lactis |
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Relations |
BioSample |
SAMN17142315 |
SRA |
SRX9721418 |
Supplementary file |
Size |
Download |
File type/resource |
GSM4984925_read_counts_KL_7029.txt.gz |
26.5 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
Raw data are available in SRA |
Processed data provided as supplementary file |
Processed data are available on Series record |
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