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Sample GSM7076016

Query DataSets for GSM7076016

Status

Public on Jan 01, 2024

Title

Mtb, S3, rep1

Sample type

SRA

Source name

bacteria

Organism

Mycobacterium tuberculosis

Characteristics

strain: Erdman
cell type: bacteria
genotype: WT
treatment: 20 uM S3

Treatment protocol

After 44 h of cultivation test compounds were added to the medium.

Growth protocol

Mtb were innoculated at an OD600 of 0.1 and cultivated for 48h at 37°C in 7H9c medium.

Extracted molecule

total RNA

Extraction protocol

Mtb were mechanically lysed by beating previously frozen cells (-80 °C) using a cell disruptor (Disruptor Genie; Scientific Industry, Bohemia, USA) and 150 - 212 µm glass beats. Lysates were then mixed with RLT lysis buffer (QIAGEN, Venlo, Netherlands) containing 1% β-mercaptoethanol (AppliChem GmbH, Darmstadt, Germany) and ethanol (Th. Geyer GmbH & Co. KG, Renningen, Germany) (3:2) and extracted using the RNeasy kit (QIAGEN, Venlo, Netherlands) according to its protocol.
RNA samples were first depleted of ribosomal RNA using the NEBNext® rRNA Depletion Kit (Bacteria) (New England Biolabs, Ipswich, USA). The following strand specific library creation was done using 1000 ng total RNA and the NEBNext® Ultra™ II Directional RNA Library Prep Kit for Illumina (New England Biolabs, Ipswich, USA). Next generation sequencing was then performed using a NovaSeq600 (Illumina, Inc, San Diego, USA) sequencer at 10 million reads per sample.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NovaSeq 6000

Description

Sample 3 and 4 were processed for relative expression compared to Samples 1 and 2 (controls)
sample name:
161586

Data processing

Sequenced reads were aligned to the M. tuberculosis reference genome (GenBank ID: NC_000962.3) using Bowtie2 version 2.3.4.1 (Langmead and Salzberg, 2012).
Known gene transcripts were quantified using featureCounts 1.5.0-p1 (Liao et al., 2014), i.e. the number of reads assigned to an annotated gene feature.
Count data normalization and differential expression testing was performed with DESeq2 version 1.34 (Love et al., 2014).
Genes were defined as differentially expressed with a fold-change >=2 and an adjusted p-value cutoff (FDR) of <=0.5. Results were visualized in R with the EnhancedVolcano version 1.12 (Kevin Blighe, 2022) and pheatmap version 1.0.12 packages.
Assembly: GenBank ID: NC_000962.3
Supplementary files format and content: featurecounts

Submission date

Mar 02, 2023

Last update date

Jan 01, 2024

Contact name

Raphael Gries

E-mail(s)

raphael.gries@uk-koeln.de

Organization name

University Hospital Cologne

Street address

Robert-Koch-Straße 21