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Sample GSM1919530

Query DataSets for GSM1919530

Status

Public on Aug 01, 2016

Title

t=0-2

Sample type

SRA

Source name

yeast

Organism

Saccharomyces cerevisiae

Characteristics

rna extraction method: hot acid phenol
strain: SK1 S.cer.
time (h in sporulation medium): 0

Extracted molecule

total RNA

Extraction protocol

RNA isolation for RNA-seq was performed as follows. RNA was isolated similar to Clarkson, B. K., Gilbert, W. V. & Doudna, J. A. Functional overlap between eIF4G isoforms in Saccharomyces cerevisiae. PLoS ONE 5, e9114 (2010). Pellets were resuspended in 1mL Acid Phenol and an equal volume of AES buffer (50mM NaAcetate pH 5.2, 10mM EDTA, 1% SDS) was added. In 2mL eppendorf tubes, samples were incubated at 65°C for 10 min with vortexing every minute. Samples were incubated on ice for 5 min and then transferred to a phaselock tube and one volume Cholrofom was added. After spinning, the top aqueous layer was transferred to a fresh phaselock tube and one volume of phenol:choloform:iaa (25:24:1) was added, tubes were spun, one volume of choloform was added, tubes were spun, and the aqueous layer was transferred to a fresh tube to be precipitated with 50uL 3M NaOAc (pH 5.5) and 550uL isopropenol. Samples were spun at max speed for 25 minutes at 4°C. The pellet was washed twice with 70% ethanol and resuspended in water.
RNA was isolated using the hot acid phenol method and DNase treated with Turbo DNase. rRNA was subtracted using the Illumina Ribo-Zero Gold rRNA Removal Kit (Yeast). RNA was then fragmented and first strand cDNA was performed. Second strand synthesis was carried out using dUTP instead of dTTP to generate strand specific libraries. Subsequently adaptors ligation, uracil digestion, and PCR were performed. All libraries were barcoded and sequenced in one NextSeq lane.
RNA-seq (Ribo-Zero, strand specific)

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NextSeq 500

Description

other (Ribo-Zero subtracted RNA)

Data processing

Reads were trimmed to 60 X 60 to be consisten with our earlier data and mapped using TopHat2, giving TopHat no annotations and allowing novel junction discovery.
A custom Bowite index was created for all splice junctions found by Tophat by concatenating the 50nt of sequence immediately before and after the junction to ensure the reads had at least a 10nt overhang on each side of the junction. Bowtie1 was run with this custom index (genome + novel splice junctions) on each end of each sequencing library separately because parried end reads would be able to map to this custom index with many 100nt fragments. Bowtie read mapping to the custom splice index was used to calculate entropy of each splice junction (see manuscript)
MISO was run on the samples to caculate Percent Spliced In (PSI).
Genome_build: SacCer3
Supplementary_files_format_and_content: TableS9.xlsx Percent Spliced In (PSI)

Submission date

Oct 27, 2015

Last update date

May 15, 2019

Contact name

Geneveive Gould

E-mail(s)

gmgould@mit.edu

Organization name

MIT

Department

Biology