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Sample GSM4038331

Query DataSets for GSM4038331

Status

Public on Nov 05, 2020

Title

Rv1720c.mutant_Glycerol_2

Sample type

SRA

Source name

Rv1720c.mutant_Glycerol_2

Organism

Mycobacterium tuberculosis

Characteristics

strain: Rv1720c mutant
genotype: mutant Mtb
time point: day4
media: Glycerol
agent: No drug

Growth protocol

The logphase culture of Mtb strains was washed with PBST twice and grown in “minimal media” (pH6.5; 0.5 g/L asparagine, 1 g/L KH2PO4, 2.5 g/L Na2HPO4, 50mg/L ferric ammonium citrate, 0.5g/L MgSO4*7H2O, 0.5mg/L CaCl2, 0.1 mg/L ZnSO4) containing either 0.1% glycerol (v/v) or 0.01% cholesterol (w/v). To assess the effect of rifampicin (RIF) on the generation of persisters, in some conditions RIF was added. RNA was isolated from the cultures at day4 using Qiagen RNaeasy Minikit according to manufacturer’s protocols (Qiagen 74104). The RNA was DNase treated using Turbo DNA free kit using manufacturer’s protocol (Thermo Fischer scientific) to remove any genomic DNA contamination. All Mycobacterial total RNAs were analyzed using an Agilent Bioanalyser (Agilent, Santa Clara, CA, USA) for quality assessment with RNA Integrity Number (RIN) range of 5.6 to 9.7 and a median of 7.5. Ribosomal RNA (rRNA) were depleted from 500ng of bacterial RNA using RiboMinus™ Bacteria transcriptome isolation kit (Invitrogen Thermo Fisher Scientific Waltham, MA, USA), according to manufacturer's protocol.

Extracted molecule

total RNA

Extraction protocol

cDNA libraries were prepared from the resultant rRNA depleted RNA and 1 ul of a 1:500 dilution of ERCC RNA Spike in Controls (Ambion® Thermo Fisher Scientific, Waltham, MA, USA) using Lexogen SENSE Total RNA-Seq Library Prep Kit (Lexogen GmnH, Vienna, Austria) according to manufacturer's protocol except with 21 PCR cycles. The length distribution of the cDNA libraries was monitored using a DNA High Sensitivity Reagent Kit on the Perkin Elmer Labchip (Perkin Elmer, Waltham, MA, USA).
All samples were subjected to an indexed paired-end sequencing run of 2x51 cycles on an Illumina HiSeq 2000 system (Illumina, San Diego, CA, USA) (16 samples/lane).

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2000

Description

RBB307
gene_read_counts_mux4168.txt.gz

Data processing

Raw reads (FASTQ files) were mapped to the M. tuberculosis H37Rv (GenBank accession AL123456) using bowtie2 using default parameters.
The read counts of genes were then counted using featureCounts v1.6.3 using the genome annotations provided in the GenBank file.
Computations were done using the R statistical language version 3.3.1.
Genome_build: M. tuberculosis H37Rv (GenBank accession AL123456)
Supplementary_files_format_and_content: Tab delimited text file with read counts for genes

Submission date

Aug 19, 2019

Last update date

Nov 05, 2020

Contact name

Kaibo Duan

E-mail(s)

duan_kaibo@immunol.a-star.edu.sg

Organization name

Singapore Immunology Network

Street address

8A Biomedical Grove, Immunos Building, Level 4