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Sample GSM2100369

Query DataSets for GSM2100369

Status

Public on Aug 12, 2016

Title

34F2R-2

Sample type

SRA

Source name

wild-type 34F3

Organism

Bacillus anthracis str. Sterne

Characteristics

strain: Sterne
genotype/variation: wild-type 34F3

Treatment protocol

For iron-depleted medium, Ristroph medium was treated with 450 μg 2,2'-dipyridyl (Sigma-Aldrich, St. Louis, MO, USA). For glucose starved condition, Ristroph medium was prepared without adding glucose. For CO2 depleted condition, Ristroph medium was prepared without adding NaHCO3 and grown under aerated atmosphere.

Growth protocol

Cells were initially grown in rich brain heart infusion (Becton Dickinson, Franklin Lakes, NJ, USA) liquid media in 15 ml conical tubes, grown at 37°C with shaking for overnight. Cells were then inoculated to Ristroph medium (3.6 g yeast extract, 0.035 g tryptophan, 0.065 g glycine, 0.025 g cystine, 0.001 g thiamine, 0.0014 g uracil, 0.0021 g adenine sulfate, 2.5 g glucose, 0.0074 g CaCl2·H2O, 0.0099 g MgSO4·H2O, 3 g K2HPO4, 0.0009 g MnSO4·H2O, 8 g NaHCO3 in 1 L DW, pH 8.0) to OD600 of 0.05, grown at 37°C under 5% CO2 atmosphere.

Extracted molecule

total RNA

Extraction protocol

RNA was extracted using RNAiso Plus (Takara, Shiga, Japan) and QIAGEN RNeasy® Mini kit (QIAGEN, Hilden, Germany) according to the manufacturer’s protocol. For RNA-Seq, RNA libraries were created from each group using the NEBNext® Ultra™ Directional RNA Library preparation kit from Illumina® (Illumina, San Diego, CA, USA). The first step in the workflow involved the removal of ribosomal RNA using the RNAMius™ Transcriptome Isolation kit (Life Technologies, Carlsbad, CA, USA). Following purification, total RNA was fragmented into small pieces using divalent cations at elevated temperature. The cleaved RNA fragments were copied into first-strand cDNA using reverse transcriptase and random primers, followed by second-strand cDNA synthesis using DNA polymerase I and RNase H. The cDNA fragments were then processed through an end-repair reaction by the addition of a single ‘A’ base, followed by ligation of the adapters. The products of these reactions were then purified and enriched by PCR to create the final cDNA library. The cDNA fragments were sequenced using the Illumina HiSeq2000 (101 cycles PE lane)
RNA libraries were prepared for sequencing using standard Illumina protocols

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2000

Data processing

Illumina CASAVA v1.8.2 software was used for basecalling.
To avoid low-quality data, we clipped and trimmed the reads using Trimmomatic (v0.33)
For the analysis of differentially expressed genes, the data of quality-checked reads for each condition were processed with the Rockhopper (version 1.30) software based on reference genome sequence (Bacillus anthracis Sterne NC_005945)
Genome_build: Bacillus anthracis Sterne NC_005945
Supplementary_files_format_and_content: tab-delimited text files including normalized read counts for each sample

Submission date

Mar 28, 2016

Last update date

May 15, 2019

Contact name

Se Kye Kim

E-mail(s)

skkim0217@gmail.com

Organization name

Hanyang University ERICA

Department

Molecular and Life Science