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Sample GSM1861090

Query DataSets for GSM1861090

Status

Public on Dec 04, 2017

Title

UT_RIP_Input Replicate 2

Sample type

SRA

Source name

Wild type yeast cells

Organism

Saccharomyces cerevisiae

Characteristics

strain: BY4741
temperature and stress: 30C
rip antibody: N/A

Growth protocol

Normal growth conditions were 30°C shaking at 200 rpm in YPAD media. Yeast cultures were grown in rich media at 30°C to an OD600 ~0.9, crosslinked with formaldehyde (1%) and then quenched with glycine. To induce oxidative stress (OS) hydrogen peroxide was added to 0.3 mM for 25 minutes prior to crosslinking.

Extracted molecule

total RNA

Extraction protocol

Cross-linked cells were harvested, then extract was made using multiple rounds of bead beating and sonication. After treatment with DNase, the IP samples were incubated with protein A-sepharose anti c-myc antibody complex. UT Input samples were treated with DNase, however they were not incubated with protein A-sepharose anti c-myc antibody complex.
Whole transcriptome cDNA libraries were synthesized using the SOLiD Total RNA-Seq Kit (PN 4445374).

Library strategy

RIP-Seq

Library source

transcriptomic

Library selection

other

Instrument model

AB SOLiD 4 System

Description

Replicate 2

Data processing

All samples were downloaded separately by barcode and mapped to the 2007 S. cerevisiae reference genome (sg7) using SHRiMP version 2.2.2. Reads were trimmed 15 bp from the 3’ end as a quality control measure. During mapping, SHRiMP2 calculated a score for each read based upon mismatches, and reads with less than 90% of the maximum possible score were filtered out (a 90% threshold is similar to allowing for 3 mismatches).
The reads per gene was tabulated after aligning reads to TSS and TTS from a published source (David et al. 2006).
To calculate enrichment, two of three replicates were processed at a time in all three combinations (i.e. rep 1 v. rep 2, rep 2 v. rep 3, and rep 1 v. rep 3) then averaged if the false discovery rate (FDR) was less than 1%. Enrichment was calculated in edgeR for each gene by dividing reads in the IP by reads in the mock IP while controlling for differences in library size among samples.
To calculate enrichment per third of a gene, reads per gene was split into reads that align to the first third, middle third and last third of each gene before calculating enrichment.
Genome_build: 2007 S. cerevisiae reference genome (sg7)
Supplementary_files_format_and_content: Tab-delimited text files that include read mapping statistics, read count per gene for each replicate or RIP-seq enrichment per gene for sample, relative to UT IP.

Submission date

Aug 25, 2015

Last update date

May 15, 2019

Contact name

Joseph C. Reese

E-mail(s)

[email protected]

Organization name

The Pennsylvania State University

Department

Biochemistry and Molecular Biology