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Sample GSM7383877

Query DataSets for GSM7383877

Status

Public on Jul 11, 2023

Title

LNA_2

Sample type

SRA

Source name

SL1344

Organism

Salmonella enterica subsp. enterica serovar Typhimurium

Characteristics

strain: SL1344
treatment: 15 min exposure to LNA

Treatment protocol

Afterward, the bacterial solution was transferred into low-binding 5 ml tubes (LABsolute) along with the 20×PNA, peptide or PPNA solution. The final concentrations were 5 μM for all test compounds. As a negative control, cells were treated with the respective volume of sterile nuclease-free water, which was used as solvent for the test compounds. After incubating the samples for 15min at 37°C, RNAprotect Bacteria (Qiagen) was added according to the manufacturer's instructions. Following a 10-min incubation, cells were pelleted at 4°C and 21,100 ×g for 20min. The supernatant was discarded and pellets were either directly used or stored at –20°C (<1 day) for subsequent bacterial RNA isolation.

Growth protocol

Bacterial overnight cultures were diluted 1:100 in fresh MHB and grown to OD600 0.5. The obtained cultures were diluted to ∼106 cfu/ml in non-cation-adjusted MHB.

Extracted molecule

total RNA

Extraction protocol

Total RNA was purified from bacterial pellets using the miRNeasy Mini kit (Qiagen) according to the protocol #3 described in Popella etal.(30) Briefly, cells were resuspended in 0.5 mg/ml lysozyme (Roth) in TE buffer (pH 8.0) and incubated for 5min. Afterwards, RLT buffer supplemented with β-mercaptoethanol, and ethanol were added according to the manufacturer's instructions. After sample loading, column wash-steps were performed according to the manual. RNA concentration was measured with a NanoDrop spectrophotometer.
Briefly, samples were treated with DNase (XXX) and quality was checked on a bioanalyzer (RNA chip XXX). Then, RNA was subjected to cDNA library preparation using the Corall kit (Lexogen) and including the depletion of ribosomal RNA (RiboCop-META kit, Lexogen) according to the manufacturer’s instructions. Afterwards, library samples were pooled in equimolar amounts and quality was verified using a bioanalyzer (DNA chip XXX). The cDNA pools were sequenced using the NextSeq 500 system (HighOutput flow cell, 400 M, 1x 75 cycle single-end; Illumina).

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NextSeq 500

Data processing

Raw RNA-Seq reads were trimmed, filtered and mapped to the SL1344 genome.
Then, BBduk was used to trim nucleobases with Phred quality scores below 10 and to remove adapter sequences.
Next, Reads were mapped against the SL1344 genome using BBMap
resulting alignments were assigned to both coding sequences and sRNAs using the featureCounts methods of the Subread package.
Assembly: Salmonella enterica subsp. enterica serovar Typhimurium str. SL1344 , Taxonomy ID: 216597
Supplementary files format and content: counttable.tsv: Count table in tab seperated format

Submission date

May 18, 2023

Last update date

Jul 11, 2023

Contact name

Jakob Jeremias Jung

E-mail(s)

jakobjung@tutanota.com

Organization name

University of Würzburg

Department

Institute of Molecular Infection Biology (IMIB)