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Sample GSM5035483

Query DataSets for GSM5035483

Status

Public on Feb 08, 2021

Title

C. diphtheriae delta-rnj2 Rep 1

Sample type

SRA

Source name

bacterial culture

Organism

Corynebacterium diphtheriae

Characteristics

strain: NCTC 13129
treatment: Gene rnj2 (DIP1463) deleted
growth phase: exponential growth

Growth protocol

Overnight cultures of C. diphtheriae NCTC 13129 and its delta-rnj2 mutant were used to inoculate fresh cultures in HIB at 30 C.

Extracted molecule

total RNA

Extraction protocol

C. diphtheriae RNA was prepared using RNeasy Mini Kits and RNase-free DNase Sets according to the manufacture’s protocol (Qiagen). Briefly, cell pellets harvested from 3 ml log-phase cultures, cultivated in triplicates per condition, were suspended into 200 μl of chilled 10 mM RNA-free Tris-HCl, pH 8.0. The suspension was added to a fast-protein tube (Q BIOgene) containing 700 μl RLT buffer (RNeasy Mini Kit, Qiagen), and cells were lysed using a Ribolyser (Hybaid). After centrifugation at 13,000 x g, RNA was purified from the supernatants accordingly. Purified RNA was treated by DNase (Qiagen) and subsequently cleaned by an RNeasy clean up kit (Qiagen). RNA quality was determined by an Agilent 2100 Bioanalyzer (Agilent Technologies) with an Agilent RNA Pico 6000 kit. RNA samples with the RNA integrity number (RIN) values of > 8.0 were stored at -800C for prior to RNA-seq analysis.
2 µg total RNA from C. diphtheriae NCTC 13129 and the delta-rnj2 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 TrueSeq 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).

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina MiSeq

Data processing

Base calling with Illumina CASAVA
quality trimming with trimmomatic v0.36 from both ends with parameters: LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 MINLEN:39
reverse complementing of R1 reads
mapping with bowtie2 2.2.7, --ff read orientation, default parameters
converting SAM to BAM with samtools 1.2
visualization and 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.

Submission date

Jan 26, 2021

Last update date

Feb 08, 2021

Contact name

Manuel Wittchen

Organization name

Bielefeld University

Department

Center for Biotechnology

Street address

Universitaetsstr. 27