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Sample GSM4852583

Query DataSets for GSM4852583

Status

Public on Oct 26, 2020

Title

H2O23

Sample type

SRA

Source name

WT bacterial culture, 0.1% oxygen

Organism

Ralstonia solanacearum

Characteristics

strain: GMI1000
genotype: WT
growth protocol: Bacterial culture, 0.1% oxygen
treatment: 100 uM hydrogen peroxide
age: 16 hpi
extraction method: Column

Treatment protocol

Column: At 12 hpi, CysNO or hydrogen peroxide were added to final concentrations of 1 mM or 100 uM, respectively, as required.

Growth protocol

Column: 50 mL of modified Van den Mooter medium (DOI: 10.1128/mBio.02471-14) with 30 mM potassium nitrate was inoculated with R. solanacearum to an initial concentration of 10^7 CFU/mL. (OD600=0.01). The cultures were then incubated without shaking at 28˚C in 0.1% oxygen for 12 hours.

Extracted molecule

total RNA

Extraction protocol

Column: After 16h total incubation, sub-samples were collected for dilution plating to determine CFU/ml and the tubes were capped and centrifuged at room temperature for 5 min at 8000 rpm. The supernatant was removed and pellets were frozen in liquid nitrogen. RNA extractions were carried out using a modified version of the Quick-RNATM MiniPrep kit (Zymo Research, Irvine, CA, USA), as follows. Pellets were resuspended in 400 µL of ice cold TE pH 8 with 1 mg/mL lysozyme, 0.25 µL Superase Inhibitor (Ambion, Austin, TX, USA), and 80 µL of 10% SDS, vortexed for 10s, transferred to a new 2 mL tube, and shaken at ~300 rpm for 2 min. 800 µL of RNA-Lysis lysis buffer was added, the tubes were vortexed again for 10 s, cleaned according to the kit manufacturer’s instructions, and eluted in 100 µL of water. Nucleic acid concentrations were estimated using a nanodrop, normalized to 200 ng/uL, and cleaned using the DNA-free DNAse kit (Invitrogen, Carlsbad, CA, USA). Samples were incubated at 37C for 1 hour, with 2 µL more of DNAse added at 30 min. After DNAse inactivation, samples were further cleaned by chloroform extraction, then precipitated overnight at -20˚C with 100 𝜇M Sodium Acetate pH 5.5 and 66 % ethanol. Samples were checked for concentration on a Nanodrop, for DNA contamination by PCR using the qRT-PCR primers serC_F/R, and for RNA integrity (RIN) using an Agilent Bioanalyzer 21000 (Agilent, Santa Clara, CA, USA). All samples had RIN values above 7.3.
NEBNext® µLtraTM Directional RNA Library Prep Kit for Illumina® (NEB, Ipswitch, MA, USA) following the manufacturer’s recommendation and starting with 3 mg of RNA per sample

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NovaSeq 6000

Data processing

Illumina CASAVA v1.8 used for basecalling
Reads were filtered for quality, removing reads with adaptor contamination, greater than 10% uncertain nucleotides, or more than 50% of nucleotides with a Qpred less than or equal to 5. Over 95% of reads were of good quality, and were mapped to the R. solanacearum GMI1000 genome (https://www.ncbi.nlm.nih.gov/assembly/GCF_000009125.1) using Bowtie2 -2.2.3
Gene expression was calculated using HTseq v0.6.1
Differential gene expression was calculated using DESeq 1.18.0. P-values calculated by DESeq were adjusted to control for the false discovery rate (FDR) using the Benjamini-Hochberg approach
Genome_build: GCA_000009125.1
Supplementary_files_format_and_content: .txt file including raw readcounts for each sample

Submission date

Oct 25, 2020

Last update date

Oct 26, 2020

Contact name

Caitilyn Allen

E-mail(s)

caitilyn.allen@wisc.edu

Phone

1 608-556-3369

Organization name

University of Wisconsin-Madison