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Sample GSM2589135

Query DataSets for GSM2589135

Status

Public on Jan 12, 2018

Title

WT_3

Sample type

SRA

Source name

Bacteria, WT

Organism

Corynebacterium diphtheriae NCTC 13129

Characteristics

strain/background: NCTC 13129
genotype/variation: wild type
growth phase: exponential

Growth protocol

Overnight cultures of C. diphtheriae NCTC 13129 and its isogenic ΔdtxR mutant were used to inoculate fresh cultures in HIB at 37 C.

Extracted molecule

total RNA

Extraction protocol

Total RNA was isolated from cells grown at exponential phase (OD600 = 0.5) using Trizol reagent (Invitrogen). The bacterial pellet obtained from 4 mL culture was resuspended in 1 mL Trizol, transferred into FastPrep Lysis Beads & Tube (MP Biomedicals) and mechanically lysed using beadbeater at a maximum speed for 20 sec 6 times. After adding 200 µL chloroform to the lysed cells followed by centrifugation at 12,000 x g for 15 min at 4 °C, the aqueous supernatant was taken and then precipitated using 500 µL isopropanol. Afterwards, crude RNA samples were treated with DNase I (Roche Diagnostics). After purification using phenol/chloroform/isoamyl alcohol (ratio 25:24:1), RNA was precipitated with ethanol. Purified total RNA pellets were dissolved in 50 µL RNase-free water.
For whole transcriptome cDNA library preparations, 2 µg total RNA from C. diphtheriae NCTC 13129 and the ΔdtxR mutant were used. Stable RNAs were depleted with the RiboZero Kit according to manufacturer's instructions (Epicentre). Afterwards, the remaining mRNA was purified using RNA MinElute columns (Qiagen) and checked for quality with the Bioanalyzer (Agilent). Fragmentation of mRNA, reverse transcription to cDNA, adenylation of 3' ends, adapter ligation and PCR amplification were performed according to TruSeq Stranded mRNA library instructions (Illumina). Prior to paired-end sequencing of the whole transcriptome cDNA libraries on an Illumina MiSeq, the quality and concentration of the libraries were checked using the Bioanalyzer (Agilent). For the primary 5'-end cDNA library, 2× 5 µg wild type RNA was used. The preparation protocol has been described previously in detail [Pfeifer-Sancar2013]. In the present study, the experimental workflow was changed at three steps. Non-primary transcripts were digested with terminator exonuclease (Epicenre) at 30 °C for 60 min and subsequently at 42 °C for 30 min. The 5' adapter ligation was performed for 60 min at 30 °C with 1 µL 60 µM adapter. After cDNA amplification, the two libraries were purified and size-selected by gel electrophoresis for fragment sizes between 100 and 1,000 bp. The cutoff of 100 bp was chosen to reduce adapter dimers in the finished library. Due to the fact that the preparation workflow involves the use of two adapters, which together have a length of 66 nt, only transcripts smaller than 40 nt are not present in the final RNA-seq data. Sequencing was performed in single-read mode with 75 nt read length for the primary 5'-end cDNA library.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina MiSeq

Description

Processed data file: List_of_differentially_transcribed_genes.xlsx

Data processing

Base calling with Illumina CASAVA.
Quality trimming with trimmomatic v0.36 from both ends (primary transcript library only from 3' end), sliding window, minimal read length 39.
Reverse complementing of R1 reads.
Mapping with bowtie2 2.2.7, --ff read orientation, default parameters.
Visualization, detection of transcription start sites, operons and gene read counting with readXplorer 2.2.3.
Analysis of differentially transcribed genes with DESeq2 in default parameters.
Genome_build: RefSeq NC_002935.1
Supplementary_files_format_and_content: List_of_differentially_transcribed_genes.xlsx contains raw counts, normalized read counts and log2foldchanges and adjusted p-values for each gene.
Supplementary_files_format_and_content: List_of_transcription_start_sites_and_operons.xlsx contains transcription start sites and operons as well as sub-operons identified with readXplorer.

Submission date

Apr 25, 2017

Last update date

May 15, 2019

Contact name

Manuel Wittchen

Organization name

Bielefeld University

Department

Center for Biotechnology

Street address

Universitaetsstr. 27