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Sample GSM5974929

Query DataSets for GSM5974929

Status

Public on Jul 01, 2022

Title

2_1_Jvpna37

Sample type

SRA

Source name

Salmonella treated with mm9 acpP PNA

Organism

Salmonella enterica subsp. enterica serovar Typhimurium str. SL1344

Characteristics

strain: SL1344
treatment: 15 min exposure to 5 uM PNA
treatment: acpP-PNA-mut-9

Treatment protocol

Bacterial overnight cultures were diluted 1:100 in fresh MHB and grown to an OD600 of 0.5. Subsequently, obtained cultures were again diluted 1:100 in fresh MHB to adjust a cell concentration of approximately 106 cfu/mL. After transferring 1.9 mL of the bacterial solution into 5 mL low-binding tubes (LABsolute), 100 µL of 20x PPNA stock solutions were added to reach a final concentration of 5 µM for all KFF-conjugated PNAs. In parallel, an equal amount of cells was treated with the respective volume of sterile nuclease-free water, which was used as solvent for the test compounds, and served as negative control. After incubating the samples for 15 min 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 xg for 20 min. The supernatant was discarded and pellets were either directly used or stored at – 20 °C (< 1 day) for subsequent bacterial RNA isolation.

Growth protocol

The strain was streaked on Luria-Bertani plates and cultured in non-cation adjusted Mueller-Hinton Broth, with aeration at 37°C and 220 rpm shaking.

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 et al. (REF). Briefly, cells were resuspended in 0.5 mg/mL lysozyme (Roth) in TE buffer (pH 8.0) and incubated for 5 min. 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

reads obtained from RNA sequencing were trimmed, filtered and mapped against the respective reference genome. The reference genome consisted of the the Salmonella enterica subsp. enterica serovar Typhimurium SL1344 (FQ312003.1) reference genome and three plasmids: pSLT_SL1344 (HE654724.1), pCol1B9_SL1344 (HE654725.1), and pRSF1010_SL1344 (HE654726.1) (Kröger et al. 2012).
Bases with a Phred quality score of <10 were trimmed and adapters were removed using BBDuk.
Next, the trimmed reads were mapped against the reference genome using BBMap (v38.84)
Alignmentts were then assigned to genomic features, including both CDSs and annotated sRNAs (Kröger et al. 2012; Hör et al. 2020) using the featureCounts method of the Subread (2.0.1) package (Liao, Smyth, and Shi 2014).
Assembly: Salmonella enterica subsp. enterica serovar Typhimurium
Supplementary files format and content: CSV format, describes the raw read counts of mRNAs and sRNAs after mapping them to the salmonella genome.

Submission date

Mar 28, 2022

Last update date

Jul 01, 2022

Contact name

Jakob Jeremias Jung

E-mail(s)

[email protected]

Organization name

University of Würzburg

Department

Institute of Molecular Infection Biology (IMIB)