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Sample GSM3466602

Query DataSets for GSM3466602

Status

Public on Oct 16, 2020

Title

WT- rep1

Sample type

SRA

Source name

Rhizobacterium

Organism

Enterobacter ludwigii

Characteristics

genotype/variation: Wild-type
strain: Strain UW5

Growth protocol

Overnight cultures from single colonies of wild-type and tyrR mutant E. cloacae were inoculated 1:100 into 3 ml fresh LB in quadruplicate and incubated at 30℃ and 250 rpm for ~2.5 hrs until reaching mid logarithmic phase of growth (OD600 = 0.6-0.9).

Extracted molecule

total RNA

Extraction protocol

A total of 0.5 ml of cell culture were transferred to a 2 ml tube containing 1 ml RNAprotect Bacteria Reagent (Qiagen), incubated 5 minutes at room temperature, and then pelleted by centrifugation. Cell pellets were resuspended with 15 mg/ml lysozyme and 15 mg/ml Proteinase K in 200 µl TE buffer to lyse cells for 10 min at 4°C. Total RNA was extracted using the RNeasy Mini Kit (Qiagen). Contaminating genomic DNA was removed by treating 5 µg total RNA in a 50 µl reaction containing 5 units Amplification Grade DNase I (Invitrogen, CA, USA) and 5 µl 10x DNase I digestion buffer and incubating at 37°C for 60 minutes. The reaction was terminated with 1 mM EDTA and heating at 70°C for 10 minutes. The DNase-treated RNA was purified using the Qiagen MinElute Cleanup Kit (Qiagen, Hilden, Germany) and eluted into 30 µl RNase-free water. DNA removal was verified by PCR. The final quality and quantity of RNA samples were measured on an Agilent Bioanalyzer-RNA 6000 Pico chip (Agilent Technologies, CA, USA).
Construction of RNA-Seq libraries and sequencing were performed by the Genome Quebec Innovation Centre (McGill, Montreal, Quebec). Ribosomal RNA was removed using the Ribo-Zero rRNA Removal Kit for Gram-Negative Bacteria (Illumina, CA, USA). The resulting enriched mRNA was used to construct a 100 bp paired-end stranded Illumina library using the TruSeq RNA Library Prep Kit v2 (Illumina). The libraries were individually barcoded and sequenced together on a single Illumina HiSeq 2000 lane.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2000

Data processing

Sequence data was inspected for overall quality using FastQC v0.11.2 (S. Andrews, http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). Low-quality reads and contaminating Illumina TruSeq adapter sequences were removed using Trimmomatic v0.3 with a sliding window size of 4 bp, cutting sequences when the average Phred score dropped below 15. Surviving paired reads were mapped to the E. cloacae UW5 reference genome (GenBank Accession number CP011798) using bwa (v0.7.9-r789) as single fragments with a maximum mismatch of 1%. Read alignments were sorted, indexed and converted to BAM format with SAMtools v0.1.19-44428cd. Sample read alignments were visualised using the Integrative Genomics Viewer v2.3.32 to ensure correct mapping of reads to genomic features. The number of reads that mapped to each genomic feature was calculated using the default parameters of HTSeq-count v 0.6.1.p1. Genes with significantly different levels of expression between wild-type and tyrR mutant strains were called using DESeq2 v1.12.4. Genes were considered to be differentially expressed when there was at least a four-fold change (i.e., |log2-fold change| > 2) in expression with a false discovery rate (FDR) threshold adjusted p-value (padj) of ≤ 0.001 as determined by the Benjamini-Hochberg correction method.
Genome_build: CP011798
Supplementary_files_format_and_content: Differential expression of all E. cloacae UW5 genes in the tyrR mutant vs. the wild-type provided in an Excel file

Submission date

Nov 13, 2018

Last update date

Oct 23, 2020

Contact name

Cheryl L. Patten

E-mail(s)

pattenc@unb.ca

Organization name

University of New Brunswick

Department

Biology