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Sample GSM2690475

Query DataSets for GSM2690475

Status

Public on Oct 04, 2017

Title

Wild Type strain harbouring empty plasmid, replicate 1

Sample type

SRA

Source name

TT01 (pBBR1MCS-5)

Organism

Photorhabdus laumondii subsp. laumondii TTO1

Characteristics

genotype/variation: wild type
overexpression: control

Treatment protocol

RNAprotect Bacteria reagent 5 min.

Growth protocol

Logarithmic culture phase in LB broth supplemented with gentamycine at 28°C.

Extracted molecule

total RNA

Extraction protocol

RNeasy miniprep QIAGEN kit.
Ribo-Zero rRNA Removal Kit Bacteria (Illumina, San Diego, CA) was used to remove ribosomal RNA from 4 µg of total RNA. For each sample, 100 ng of rRNA-depleted RNA was used to construct sequencing libraries using Illumina’s TruSeq Stranded mRNA Sample Prep Kit (Low throughput). The mRNA was chemically fragmented. The first cDNA strand was generated by reverse transcription with random hexamer primers, SuperScript IV Reverse Transcriptase (Life Technologies), Actinomycine D. The second strand cDNA was synthesized by replacing dTTP with dUTP. A single ‘‘A’’ nucleotide was added to the 3' end and ligation was carried out with Illumina’s indexed adapters. After 15 cycles of PCR, libraries were validated on a Fragment Analyzer (AATI, Ankeny, IA) and quantified with a KAPA qPCR kit.
On a sequencing lane of a flowcell V4, nine libraries were pooled in equal proportions, denatured with NaOH and diluted to 8 pM before clustering. Clustering and 50nt single read sequencing were performed according to the manufacturer’s instructions.
Cluster formation, primer hybridisation and single end-read 50 cycles sequencing were performed on cBot and HiSeq2500 (Illumina, San Diego, CA) respectively.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2500

Description

pBB_A
P. luminescens TT01
Processed data file: raw_reads_count.txt
Processed data file: normalized_reads_count_TT01(pBBR1MCS-5)_versus_TT01(pBB-Dam).txt

Data processing

Image analyses and basecalling were performed using the Illumina HiSeq Control Software and Real-Time Analysis component.
Demultiplexing was performed using Illumina's conversion software (bcl2fastq 2.17).
The quality of the data was assessed using FastQC from the Babraham Institute and the Illumina software SAV (Sequencing Analysis Viewer). Potential contaminants were investigated with the FastQ Screen software from the Babraham Institute.
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 Photorhabdus luminescens TT01 genome sequence 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: Photorhabdus luminescens TT01 (NC_005126.1)
Supplementary_files_format_and_content: raw_reads_count.txt: Tab-delimited text file includes raw number of reads mapped to individual genes of the Photorhabdus luminescens TT01 genome, for all samples.
Supplementary_files_format_and_content: normalized_reads_counts_TT01(pBBR1MCS-5)_versus_TT01(pBB-Dam).txt: Tab-delimited text file includes normalized reads count compute by DESeq package for the diferential expression between the two conditions : TT01 (pBBR1MCS-5) versus TT01 (pBB-Dam).

Submission date

Jun 29, 2017

Last update date

May 15, 2019

Contact name

Anne Lanois

E-mail(s)

anne.lanois-nouri@umontpellier.fr

Organization name

INRA

Street address

75 rue du Truel CC54