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Sample GSM2781067

Query DataSets for GSM2781067

Status

Public on Jan 23, 2022

Title

delta-VirR BHI 2

Sample type

SRA

Source name

delta-VirR BHI

Organism

Listeria monocytogenes

Characteristics

strain: BUG2952
genotype/variation: delta-VirR

Treatment protocol

the mutant and the wild-type strains were grown in BHI to exponential phase (OD600 0.5-0.7), pelleted and stored at -80°C till further RNA isolation. Three biological replicates (individual colonies) of wild type and mutant strains were grown on different days.

Growth protocol

the bacteria were grown in BHI (Difco) from an overnight preculture.

Extracted molecule

total RNA

Extraction protocol

Bacterial pellets from 10 ml cultures were re-suspended in 500 ul of 20 mM Tris-HCl buffer pH 8.0 containing 50 mM EDTA. The suspensions were added to 500 ul of acid-saturated phenol:chloroform, pH 4.5 (with isoamyl alcohol, 125:24:1) (Ambion) and transferred to 2 ml Lysing Matrix Tubes (MP Biomedicals, 6911). Bacteria were mechanically lysed in a FastPrep apparatus (two cycles of 45 sec, speed 6.0 with one minute pause on ice). Subsequently tubes were centrifuged at 4°C at maximum speed for 15 min in a tabletop centrifuge. Lysates were transferred to new 2ml Eppendorf tubes containing 500 ul phenol and 500 ul chloroform, vigorously vortexed and centrifuged at 4°C at maximum speed for 15 min. The upper aqueous phase was transferred to new 1.5 ml tubes and RNA was precipitated with 500 ul isopropanol in the presence of 0.3 M sodium acetate pH 5.5 for 5 min at -80°C followed by 15 min at -20°C. RNA was pelleted by centrifugation at maximum speed for 30 min at 4°C. Pellets were washed with 75% ethanol, air dried and re-suspended in 50-70 ul water.
The input total RNAs had RNA integrity numbers of typically 9.8-10 as defined with 2100 Bioanalyser instrument (Agilent). Total RNA were diluted to 200 ng/ml and treated with TURBO DNase (Ambion) for 15 min at 37°C. DNase was removed with DNase inactivation reagent according to manufacturer’s instructions. 4 mg of TURBO DNase-treated RNA was depleted for ribosomal RNA with Ribo-Zero™ Magnetic kit (Epicentre). Zymo-Spin IC columns were used for RNA purification. RNA was subsequently fragmented for 5 min at 70°C using Ambion Fragmentation Reagent and purified with Zymo-Spin columns. Fragmented RNAs were treated with Antarctic phosphatase (New England Biolabs) for 30 min at 37°C followed by 5 min at 65°C for phosphatase inactivation. 5’-ends of the fragmented RNAs were then phosphorylated with polynucleotide kinase (New England Biolabs) for 60 min at 37°C in the presence of RNaseOUT™ ribonuclease inhibitor (Thermo Fischer Scientific). cDNA libraries were prepared using NEBNext® Multiplex Small RNA Library Prep Set for Illumina® (New England Biolabs) according to manufacturers’ instructions. Libraries were purified with AMPure XP beads (Agencourt) and checked for quality with Bioanalyzer DNA High Sensitivity Chips (Agilent). Sequencing was performed on a MiSeq (Illumina) in a multiplexed single read using a MiSeq Reagent Kit v3 (Illumina). Raw sequence files (fastq) were cleaned of 3' adapter sequences, and low quality (cutoff 28) and short sequence (15 nt) reads were removed using AdapterRemoval v1.5.2 software (51). Reads were aligned against the reference genome (NC_003210) with bowtie v1.0.0 (52) using default parameters. Typically 94% reads were aligned. Reads were counted with HTSeq v0.6.0 and normalized to reads per million. Differential expression analysis was performed with DeSeq2 software package
Directional libraries were constructed with the use of a custom ligation based protocol (adapted from NEBNext® Small RNA Library Prep Set for Illumina®, #E7330)

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2000

Description

delta-VirR BHI 2

Data processing

Raw sequence files (fastq) were cleaned of 3' adapter sequences, and low quality (cutoff 28) and short sequence (15 nt) reads were removed using AdapterRemoval v1.5.2 software
Reads were aligned against the reference genome (NC_003210) with bowtie v1.0.0 using default parameters
Raw counts were generated with HTSeq v0.6.0
Differential expression analysis was performed with DeSeq2 software package with default parameters
Genome_build: NC_003210
Supplementary_files_format_and_content: WT_vs_VirR.complete.txt, counts, fold-change

Submission date

Sep 12, 2017

Last update date

Jan 23, 2022

Contact name

Pascale Cossart

Organization name

Institut Pasteur

Lab

UIBC

Street address

25 rue du Dr Roux