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Sample GSM4485607

Query DataSets for GSM4485607

Status

Public on Apr 20, 2020

Title

PAO1 clpV3-KO rep2

Sample type

SRA

Source name

PAO1(ΔclpV3)

Organism

Pseudomonas aeruginosa PAO1

Characteristics

strain: PAO1
genotype/variation: clpV3 deletion mutant

Treatment protocol

grow until mid-exponential phase in LB

Growth protocol

liquid culture in LB (Luria-Bertani) medium

Extracted molecule

total RNA

Extraction protocol

Strains were grown overnight at 37 °C followed by sub-culturing into fresh medium and grown to mid-exponential phase. Total RNA was extracted by TRIzol-based method (Life Technologies, CA, USA). In brief, the cultures were centrifuged at 12, 000 g for 5 minutes. The supernatant was discarded, cell pellets were resuspended in 1 ml of TRIzol and then incubated at room temperature for 5 minutes to permit complete dissociation of nucleoproteins complex. Followed by adding 0.2 ml chloroform into the tube and mix well, the samples were centrifuged (12, 000 g at 4 °C for 15 minutes) to form three layers. The upper aqueous phase was transferred to a fresh tube and added by 0.5 ml cold isopropanol to precipitate RNA. After centrifugation, the supernatant was removed, and the pellet was suspended in 1 ml 75% ethanol. Samples were centrifuged and the supernatant was discarded, air-dried RNA pellet was resolved in 50 µL RNase-free Water.
RNA integrity was measured using Bioanalyzer 2100 (Agilent, Santa Clara, CA) and rRNA was removed from 1 mg of total RNA with Ribo-Zero Magnetic Gold Kit (Epicentre Biotechnologies, Madison, WI, USA). To construct the RNA-Seq library, TruSeq RNA Sample Prep Kit v2 (Illumina, San Diego, CA, USA) was used. rRNA-depleted RNA was fragmented into small pieces using Elute Prime Fragment Mix. First-strand cDNA was synthesized with First Strand Master Mix and Super Script II reverse transcriptase (Invitrogen, Carlsbad, CA, USA). Following purification by Agencourt RNAClean XP beads (Beckman Coulter, CA, USA), the second-strand cDNA library was synthesized using Second Strand Master Mix and dATP, dGTP, dCTP, dUTP mix. Purified fragmented cDNA was end repaired (30 minutes at 37 °C) prior to ligating sequencing adapters. Amplified RNA-Seq libraries were purified by using AMPureXP Beads.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2500

Data processing

After RNA-Sequencing, the obtained raw data were filtered according to the following standards: 1) removing reads with ≥ 10 % unidentified nucleotides (N); 2) removing reads with > 50 % bases having Phred quality scores of ≤ 20; 3) removing reads aligned to the barcode adapter using FASTP (https://github.com/OpenGene/fastp).
Quality trimmed reads were aligned using Bowtie2 (Langmead and Salzberg, 2012) (version 2.2.8) to the P. aeruginosa PAO1 reference genome to identify known genes and calculated gene expression by RSEM (Li and Dewey, 2011).
The gene expression level was calculated and further normalized by using the fragments per kb of transcript per million (FPKM) mapped reads method to eliminate the influence of different gene lengths and amount of sequencing data on the calculation of gene expression.
The edgeR package (http://www.r-project.org/) was used to identify differentially expressed genes (DEGs) across samples with fold changes ≥ 2 and a false discovery rate-adjusted P (q value) < 0.05.
Go terms and KEGG pathway were defined as being significantly enriched when the q value ≤ 0.05.
Genome_build: Pseudomonas aeruginosa PAO1

Submission date

Apr 19, 2020

Last update date

Apr 22, 2020

Contact name

Kangmin Duan

E-mail(s)

[email protected]

Organization name

University of Manitoba

Street address

66 Chancellors Cir