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Sample GSM2668130

Query DataSets for GSM2668130

Status

Public on Jun 04, 2019

Title

WT-YPD_rep2

Sample type

SRA

Source name

WT-YPD

Organism

Saccharomyces cerevisiae

Characteristics

strain background: BY4741 (S288C, MATa)
genotype/variation: WT
media condition: YPD
starting cell density: OD600 = 0.2
amount of growth/incubation: 3-4 population doublings
growth phase: exponential
cell type: Budding yeast cells

Growth protocol

Following an overnight growth, cells were diluted in the various listed media conditions and cell culture densities. Cells were then grown/incubated for the indicated amounts of time. Note that due to different growth rates, time in culture was often different between strains.

Extracted molecule

total RNA

Extraction protocol

Total RNA was extracted by the hot acid-phenol method (Schmitt et al., 1990), and further purified by phenol-chloroform extraction. Following extraction and ethanol precipitation, total RNA was DNAse-treated with Turbo DNAse (ThermoFisher) according to the manufacturer's protocol.
polyA+ RNA libraries were prepared using either the KAPA Stranded mRNA-seq library prep kit or the Illumina TruSeq Stranded mRNA Library Kit for NeoPrep, as indicated. Libraries were prepared according to the manufacturer's protocols. Both library preparation procedures first isolate polyA+ mRNAs using oligo-dT beads. Isolated polyA+ RNAs are then chemically fragmented, and random primers are used in the first cDNA reverse transcription step. Note that while both kits prepare "first strand" cDNA libraries, the strand-specific information was not used for data analysis in our study. However, that information can be extracted from the FASTQ files. Average library insert sizes were ~170 bp. High throughput sequencing was performed on Illumina’s HiSeq 4000 system, with single-end 50 bp insert reads, and dedicated index reads.
Library prep: KAPA Stranded mRNA-seq library prep kit

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 4000

Data processing

Sequence reads were aligned to the R64-1-1 S288C reference genome assembly (sacCer3) using Tophat 2.0.9. The “-g 1” parameter was used to limit the number of alignments for each read to one, the top-scoring alignment.
Gene expression values, in reads per kilobase per million mapped reads (RPKMs), for 6692 annotated open reading frames were calculated using SAMMate 2.7.4. (Xu et al., 2011). In calculating RPKM values, only reads that align to annotated exons were considered. Reads aligning to introns or intergenic regions were not counted in normalizing the read counts to the total number of mapped reads. Read counts and RPKM values for all 6692 ORFs can be found in GEOoscarcampos_All_ORFs_RPKMs_Jun2017.txt.
Many of the 6692 annotated ORFs have no attributed function, localization, mutant phenotypes or interactions and are often labeled as “dubious” or “putative” in the Saccharomyces Genome Database (SGD). Furthermore, these putative ORFs typically have low expression values. We removed 1648 such ORFs from further analysis, and used the remaining 5044 ORFs for comparisons between groups. RPKM values for 5044 ORFs can be found in GEOoscarcampos_5044_ORFs_RPKMs_Jun2017.txt.
Genome_build: R64-1-1 S288C reference genome assembly (sacCer3)
Supplementary_files_format_and_content: GEOoscarcampos_All_ORFs_RPKMs_Jun2017.txt contains read counts and RPKM values in tab-delimited format. Each pair of columns corresponds to different samples and each row is an ORF. The first 4 columns contain ORF information. GEOoscarcampos_5044_ORFs_RPKMs_Jun2017.txt contains only RPKM values in tab-delimited format. Each column corresponds to different samples and each row is an ORF. The first 5 columns contain ORF information.

Submission date

Jun 14, 2017

Last update date

Jun 04, 2019

Contact name

Oscar Antonio Campos

Organization name

University of California, Los Angeles

Department

Biological Chemistry

Lab

Kurdistani