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Sample GSM2108275

Query DataSets for GSM2108275

Status

Public on Jan 26, 2017

Title

chrr_merr_KO_Dark_rep1

Sample type

SRA

Source name

chrr_merr_KO_Dark

Organism

Vibrio cholerae O1 biovar El Tor str. N16961

Characteristics

strain: O1 biovar eltor str. N16961
genotype: delta_VCA0056, delta_VC2301 (MerR,ChrR)
light status: dark

Treatment protocol

For blue-light experiments, V.cholerae cells were inoculated into 5 mL streptomycin supplemented LB and grown overnight under complete darkness by covering the tubes with aluminum foil. Then, dark kept cells were diluted (1:50) in 15 mL streptomycin supplemented LB and grown until OD600=0.8-1.0. After that, cells were harvested by centrifugation at 4000 rpm for 5 minutes, washed once with PBS and resuspended in 15 mL PBS buffer . Cells were then exposed to blue-light (50 µmole m-2s-1) for 45 minutes.

Growth protocol

V. cholerae cultures were grown in Luria-Bertani (LB) broth (1% tryptone, 0.5% yeast extract, and 1% NaCl) with supplemented streptomycin (100 μg/ml) at 37°C with agitation (250 rpm).

Extracted molecule

total RNA

Extraction protocol

Total RNA was extracted from OD600=1.0, 500 µl cell culture by using TRIzol reagent according to the manufacturer’s instructions.
After quality and quantity measurements of RNA by using 2100 BioAnalyzer, RNA was treated with RNase-free DNase I at a concentration of 1 U/µg to remove residual genomic DNA. In order to remove the rRNA, 2.5 µg total RNA was treated with Ribo-Zero™ bacterial rRNA removal kit following the manufacturer instructions. The rRNA depleted RNA pellets obtained after the ethanol precipitation step was re-suspended in 10 μl of RNase free water. After that, RNA samples were fragmented using fragmentation buffer by using TruSeq mRNA Sample Preparation Kit . The cleaved short RNA fragments were used for the first-strand cDNA synthesis using random hexamer-primer, and then the second strand was synthesized by using DNA polymerase I and RNase H. The double strand cDNAs were purified with AMPure XP beads and eluted with resuspension buffer followed by 3’end adenine nucleotide addition. Finally, sequencing adaptors were ligated to the fragments and cDNA fragments were enriched by PCR amplification. Enriched cDNA libraries were used for cluster generation and sequencing. Paired-end sequencing of the two cDNA libraries (dark and blue-light exposed) of each wild type and mutant V. cholerae cells was performed using the Illumina MiSeq sequencing platform. All sequence data are PE 2x75 bp. Image processing, base calling, and quality calue calculation were performed by the Illumina data processing pipeline (v1.5). High quality reads were saved in FASTQ format.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina MiSeq

Data processing

Illumina data processing pipeline (v1.5) was used for base calling.
FastQC (v0.11.5) was used to evaluate the initial quality of the raw reads.
The following steps were performed to obtain high quality clean reads using Trimmomatic (v0.32) tool: (1) removing the reads with adaptor contamination; (2) filtering the low-quality reads with ambiguous sequences ‘N’; (3) removing the low-quality bases (quality score < Q30).
Data resulting from the above RNA-seq experiments was analyzed with the tool Rockhopper. Reads were aligned to the V. cholera O1 biovar El Tor str. N16961 genome with the parameters: minimum seed length 0.33 and allowed mismatches 15%. Following alignment of the sequencing reads to the genome, reads from each experiment were normalized by upper quartile normalization [17]. A Pearson correlation analysis was conducted to obtain the transcript-level R2 between replicates.
We used the q-value < 0.01 (FDR) and the absolute value of |Fold change| ≥ 2 as the threshold to judge the significant differences of gene expression.
Genome_build: ASM674v1
Supplementary_files_format_and_content: Microsoft Office Open XML Spreadsheet with abundance measurements

Submission date

Apr 04, 2016

Last update date

May 15, 2019

Contact name

mehmet tardu

E-mail(s)

mtardu@umich.edu

Organization name

University of Michigan

Department

Chemistry