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Sample GSM1354477

Query DataSets for GSM1354477

Status

Public on Apr 08, 2014

Title

RD19-NI+TEX

Sample type

SRA

Source name

upper sand layers of the Sahara

Organism

Deinococcus deserti

Characteristics

strain: RD19
genotype/variation: wild-type
treatment: non-irradiated, TEX-treated

Treatment protocol

For RNAseq, a 100 ml culture of RD19 was grown to exponential phase (OD600 0.5), and then divided in two. One part (45 ml) was exposed to 1 kGy gamma irradiation at room temperature (23 Gy/min, 60Co source; CEA/Cadarache, France), and then recovered for 30 min. The other part (45 ml) was not irradiated but otherwise treated in the same manner. After recovery, 15 ml of each culture was added to RNAprotect Bacteria Reagent (Qiagen) to stabilize RNA, following the instructions of the manufacturer.

Growth protocol

D. deserti strain RD19 is a spontaneous streptomycin resistant derivative of the wild-type strain VCD115. It is routinely grown at 30°C with aeration in tenfold diluted tryptic soy broth supplemented with trace elements

Extracted molecule

total RNA

Extraction protocol

RNAprotect-treated cells were centrifuged and cell pellets stored at -80°C. Cell pellets were pretreated with lysozyme and proteinase K. Total RNAs were isolated using the mirVana RNA isolation kit (Ambion) including DNase treatment.
From each RNA preparation (i.e. non-irradiated and irradiated cells), two cDNA syntheses were carried out, one with and one without Terminator exonuclease (TEX, Epicentre) treatment. cf SAMPLES
RNA was incubated with TEX (Terminator exonuclease (TEX, Epicentre)), which specifically degrades RNA species that carry a 5' mono-phosphate (5'P). The exonuclease resistant RNA (primary transcripts with 5'PPP) was poly(A)-tailed using poly(A) polymerase and treated with TAP (tobacco acid pyrophosphatase), which degrades 5'PPP to 5'P. Then an RNA adapter was ligated to the 5'P of the “de-capped” RNA. First-strand cDNA synthesis was performed using an oligo(dT)-adapter primer and M-MLV reverse transcriptase. The resulting cDNAs were PCR-amplified to about 30 ng/μl using a high fidelity DNA polymerase. The cDNAs were purified using the Agencourt AMPure XP kit (Beckman Coulter Genomics) and analyzed by capillary electrophoresis. The primers used for PCR amplification were designed for TruSeq sequencing according to the instructions of Illumina, with the 3’-sequencing adapter containing a barcode specific for each library. The +TEX cDNA samples were pooled at approximately equimolar amounts and the cDNA pool was used for sequencing without further treatment. The pools were sequenced on a Illumina HiSeq 2000 machine (read length: 100 bp).

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2000

Description

Sample 3
processed data file: geneTSScount_RD19+TEX.txt
processed data file: orphanTSScount_RD19+TEX.txt
barcode: ATCACG

Data processing

In a first step, the RNA-Seq data quality was assessed by including option like reads trimming or merging/split paired-end reads.
In a second step, reads were mapped onto the D. deserti VCD115 genome sequence (GenBank accession numbers CP001114, CP001115, CP001116 and CP001117 for the chromosome and plasmids P1, P2 and P3, respectively) using the SSAHA2 package (Ning et al, 2001) that combines the SSAHA searching algorithm (sequence information is encoded in a perfect hash function) aiming at identifying regions of high similarity, and the cross-match sequence alignment program (Ewing et al, 1998), which aligns these regions using a banded Smith-Waterman-Gotoh algorithm. An alignment score equal to at least half of the read is required for a hit to be retained.
To lower false positives discovery rate, the SAMtools (v.0.1.8) were then used to extract reliable alignments from SAM formatted files.
The number of reads matching each genomic object harbored by the reference genome was subsequently computed with the Bioconductor-GenomicFeatures package.
The Bioconductor-DESeq package with default parameters was used to analyze raw counts data and test for differential expression between conditions.
Genome_build: Deinococcus deserti VCD115
Supplementary_files_format_and_content: Tab-delimited text file include raw number of reads mapped to individual genes for RD19-NI and RD19-IR experiments.
Supplementary_files_format_and_content: Tab-delimited text file include differential expression analysis from RD19 IR compared to RD19 NI.
Supplementary_files_format_and_content: Tab-delimited text file include reads first positions counts at gene TSSs for RD19-NI+TEX and RD19-IR+TEX experiments.
Supplementary_files_format_and_content: Tab-delimited text file include reads first positions counts at orphan TSSs for RD19-NI+TEX and RD19-IR+TEX experiments.

Submission date

Mar 20, 2014

Last update date

May 15, 2019

Contact name

Arjan de Groot

E-mail(s)

[email protected]

Organization name

CEA

Department

BIAM-SBVME