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Sample GSM2398350

Query DataSets for GSM2398350

Status

Public on Jan 01, 2018

Title

cerevisiae upf1D untreated replicate B

Sample type

SRA

Source name

yeast culture

Organism

Saccharomyces cerevisiae

Characteristics

cell type: haploid, vegetative
strain: BY4741, upf1::KanMX4

Treatment protocol

Untreated cells were harvested by centrifugation after growth, or after growth plus 1 hour treatment with 200 uM rapamycin as per Munding et al. (2013) Mol. Cell 51: 338-48.

Growth protocol

Yeast were grown in YEPD to early log phase (OD600 about 0.25 to 0.3) at 30°C (S. cerevisiae) or 26°C (S. mikatae, S. bayanus)

Extracted molecule

total RNA

Extraction protocol

RNA was isolated as described in Rio et al. (RNA: A Laboratory Manual, CSH Press)
RNA was treated using the RiboZero kit to remove ribosomal RNAs and then RNA libraries were prepared for sequencing using standard Illumina protocols

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 4000

Description

cereviu_rapa00_jt904

Data processing

RNAseq 1a: the 100x100bp pairedEnd reads for cerevisiae samples were not trimmed.
RNAseq 1b: the 51x51bp pairedEnd reads for mikatae and bayanus samples were not trimmed.
RNAseq 2: reads mapping (by bowtie2) to cerevisiae rRNA, tRNA elements defined by Ensembl, or to Ty elements defined in SGD, were discarded.
RNAseq 3a: cerevisiae reads were mapped to sacCer3 by STAR 2.4.1d, with "twopass" mode.
RNAseq 3b: mikatae reads were mapped to sacMik2 by STAR 2.4.0h with "twopass" mode
RNAseq 3c: bayanus reads were mapped to sacBay2 by STAR 2.4.0h with "twopass" mode
RNAseq 4: duplicate read mappings of the same start and end position were discarded
Ratios involving three coverage numbers were generated for each of the samples and each splice junction. A splice junction coverage was the number of reads that contained the splice junction. The intron coverage was generated from the coverage data using the average across the whole intron. When introns overlapped, a minimal length intron was used such that start of the intron was the maximal start of any of the overlapping introns and the end of the minimal length intron was the minimal end of any overlapping intron. Exon2 coverage was the average coverage for 100 bases of the following exon. Log2-transformed ratios were calculated for the three comparisons: intron/exon2, junction/exon2, intron/junction. Final scores were generated by subtracting the timepoint zero score for each timepoint. Separately intron splice sites and branch points are classified. Given an intron position the splice sites are extracted with conservation scores and a most likely branch point is found using several heuristics. The likely branch points are prioritized 1.)ACTAA, 2.)PYTPAYP, 3.)YTPAY. It must be 45 or more bases away from the 5' splice site and the one closest to the 3' splice site is chosen. Conservation is then calculated around the adenosine branch point. Details and scripts are at: http://exon.ucsc.edu/intron_bp_generator/
Genome_build: sacCer3 (UCSC Apr 2011 )
Genome_build: sacMik2 (sequences obtained in Feb 2015 from the Saccharomyces sensu stricto database)
Genome_build: sacBay2 (sequences obtained in Feb 2015 from the Saccharomyces sensu stricto database)
Supplementary_files_format_and_content: Intron_master_list.xls: junction coordinates and counts, splice site identification and conservation, bp prediciton, intron and exon2 coverage and log2 ratios, change in log2 ratios after rapamycin treatment, splicing efficiency
Supplementary_files_format_and_content: Deseq_Log2_0vs60.xls: DEseq log2 ratios of expression changes in s. cerevisiae after treatment with rapamycin.

Submission date

Nov 21, 2016

Last update date

May 15, 2019

Contact name

Jason Talkish

E-mail(s)

jtalkish@ucsc.edu

Phone

831-459-4917

Organization name

University of California, Santa Cruz