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Sample GSM3530868

Query DataSets for GSM3530868

Status

Public on Nov 02, 2020

Title

PA14 ∆rclR 2

Sample type

SRA

Source name

Bacterial cells

Organism

Pseudomonas aeruginosa

Characteristics

description: Pseudomonas aeruginosa UCBPP-PA14 strain
genotype: delta_rclR
background strain: UCBPP-PA14

Treatment protocol

The concentration of HOCl required was prepared by diluting sodium hypochlorite (NaOCl) solution with 10-15% available chlorine (Sigma) directly into LB. This was further diluted into the exponential phase cultures to a final concentration of 2.2 mM HOCl (0.015-0.0225% chlorine), and cultures were incubated for 20 minutes. HOSCN was prepared enzymatically by combining glucose, glucose oxidase (GO), potassium thiocyanate (KSCN) and lactoperoxidase (LPO) directly in LB. This was diluted in the exponential phase cultures to a final concentration of 0.8 mM HOSCN (0.01% glucose, 0.5 U/ml GO, 0.75 mM KSCN and 3 U/ml LPO), and cultures were incubated for 20 minutes.

Growth protocol

PA14 WT and ∆rclR strains were grown overnight in LB broth at 37◦C on a shaking platform (two biological replicates of each strain). The four cultures were subcultured 1:50 in LB and grown to mid-exponential phase (37◦C, shaking, for 2.5 hours). These exponential phase cultures were subcultured, three separate times, 1:50 in LB and grown again for 2.5 hours. At this point, either HOCl was added to a final concentration of 2.2 mM, or HOSCN was added to a final concentration of 0.8 mM or nothing was added (untreated control). Cultures were incubated for a further 20 minutes before recording OD600, harvesting cells and extracting RNA.

Extracted molecule

total RNA

Extraction protocol

After 20 minute incubation with HOCl or HOSCN, cells were harvested, washed and RNA preserved by addition of RNAlater (Ambion). Bacteria were lysed with lysozyme and proteinase K buffer and total RNA extracted using the RNeasy Protect Bacteria Mini Kit (Qiagen), according to the manufacturer’s instructions. RNA samples were DNase treated using a TURBO DNA-free kit (Ambion), according to the manufacturer’s instructions, prior to sending RNA on dry ice to vertis Biotechnologie AG for preparation of cDNA libraries and Illumina sequencing.
rRNA was removed using the bacterial Ribo-Zero rRNA Removal Kit (Illumina), and then RNA fragmented by ultrasound. An oligonucleotide adapter was ligated to the 3’ end of the RNA and first-strand cDNA synthesis carried out with M-MLV reverse transcriptase. The cDNA was purified and a 5’ Illumina TruSeq sequencing adapter (with a TruSeq barcode sequence) was ligated to the 3’ end of the antisense cDNA. PCR amplification of the cDNA to about 10-20 ng/µl was carried out with a high-fidelity DNA polymerase for 11 or 12 cycles. The cDNA was purified and then pooled and size fractionated in the range of 200-500 bp. The cDNA pool was single-read sequenced on an Illumina NextSeq 500 system using a 75 bp read length.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NextSeq 500

Data processing

Reads were mapped using bowtie2 and aligned to the reference genome Pseudomonas aeruginosa UCBPP-PA14 (NC_008463.1)
Expression values in Reads Per Kilobase per Million mapped reads (RPKM) were calculated using FeatureCounts
RPKM values were normalised against the OD600 of the samples
Log2 fold change of WT+HOCl vs. WT untreated, WT+HOSCN vs. WT untreated, WT+HOCl vs. ∆rclR+HOCl and WT+HOSCN vs. ∆rclR+HOSCN was calculated
Genome_build: Pseudomonas aeruginosa UCBPP-PA14, complete genome (NC_008463.1)
Supplementary_files_format_and_content: Excel spreadsheet containing tab 1: expression data reads, RPKM values; tab 2: RPKM OD normalised values; tab 3: log2 fold change values of WT+HOCl vs. WT untreated, WT+HOSCN vs. WT untreated, WT+HOCl vs. ∆rclR+HOCl and WT+HOSCN vs. ∆rclR+HOSCN

Submission date

Dec 26, 2018

Last update date

Nov 02, 2020

Contact name

Huw David Williams

E-mail(s)

[email protected]

Organization name

Imperial College London

Department

Department of Life Sciences