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Sample GSM1115753

Query DataSets for GSM1115753

Status

Public on Dec 15, 2013

Title

Denatured (95C) rep1

Sample type

SRA

Source name

Denatured_95C

Organism

Saccharomyces cerevisiae

Characteristics

strain: BY4741
culture condition: Denatured_95C

Treatment protocol

For in vivo samples, DMS was added directly to the growing yeast culture at 2-4% for 2-3 min. 25%BME, 30% isoamyl alcholol was used to quench the DMS. Cells were lyzed with SDS and RNA was isolated with hot acid phenol. For in vitro samples, total RNA was extracted from yeast cells , polyA mRNA was selected, denatured at 95C, refolded in RNA folding buffer for 30min at 30C, and then probed with DMS at 2-5% for 3-5min.

Growth protocol

WT yeast were grown to exponential phase at 30C

Extracted molecule

polyA RNA

Extraction protocol

Total RNA was extracted using hot acid phenol. mRNA was extracted with Dynal beads(invitrogen)
mRNA was randomly fragmented using Ambion fragmentatin reagents, followed by PNK, and ligation using T4 K227Q truncated RNA ligase. Superscript III was used for reverse trancription , and circ ligase (epicentre) for circularization. Illumina adapters were introduced by 8-10 cycles of PCR.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2000

Description

Sample 5
processed data files: Feb13_DenaturedAll_1_15_Plus.wig,
Feb13_DenaturedAll_1_15_MINUS.wig,
ACden1_LateJune12res_sc-rrna.align18sRNAnoMiss.wig,
r25sDMS_ACden1_LateJune12res_sc-rrna.align.wigFiveCount.wig

Data processing

Illumina Casava1.7 software used for basecalling.
Alignments were performed with bowtie using the first 25 nt of each read
Reads were first aligned to the ribosomal RNA and the unaligned reads were then aligned to the genome
reads were filtered for unique map to the genome and no mismatches
reads were filtered for reverse transcriptase stops at As and Cs only
Genome_build: Saccharomyces cerevisiae assembly R61 (UCSC: sacCer2).
Supplementary_files_format_and_content: The position number directly indicates the DMS modified position starting at 1. Raw reads for each replicates were combined together in wiggle format. Separate files are provided for positive and negative strand maching reads.
Supplementary_files_format_and_content: For the ribosomal RNA, processed wig files are aligned to the ribosomal RDN locus (RDN18 is 18S rRNA and RDN25 is 25S rRNA).

Submission date

Apr 05, 2013

Last update date

May 15, 2019

Contact name

Silvi Rouskin

E-mail(s)

srouskin@wi.mit.edu

Organization name

Whitehead Institute

Street address

455 Main Street