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Sample GSM2520772

Query DataSets for GSM2520772

Status

Public on Apr 03, 2017

Title

RD42-NI

Sample type

SRA

Source name

upper sand layers of the Sahara

Organism

Deinococcus deserti

Characteristics

strain: RD42
genotype/variation: irrE mutant
exposed to: none (non-irradiated)

Treatment protocol

For RNAseq, a 100 ml culture of RD42 or RD62 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 strains are 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), one cDNA synthesis was carried out. RNA was directly poly(A)-tailed using poly(A) polymerase, followed by TAP treatment. An RNA adapter was then ligated to the 5'P of the total RNA samples. First-strand cDNA synthesis was performed using an oligo(dT)-adapter primer and M-MLV H- reverse transcriptase. The resulting cDNAs were PCR-amplified to about 60-90 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. For Illumina sequencing, the samples were pooled at approximately equimolar amounts and size fractionated in the range between 250 – 500 bp on Agarose Gel. 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

processed data file:
differentialExpression_RD42.txt

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:
readCountGenes_RD42_RD62.txt: raw number of reads mapped to individual genes for RD42-NI, RD42-IR, RD62-NI and RD62-IR experiments.
differentialExpression_RD42.txt: differential expression analysis from RD42-IR compared to RD42-NI
differentialExpression_RD62.txt: differential expression analysis from RD62-IR compared to RD62-NI

Submission date

Mar 03, 2017

Last update date

May 15, 2019

Contact name

Arjan de Groot

E-mail(s)

[email protected]

Organization name

CEA

Department

BIAM-SBVME