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Sample GSM7498436

Query DataSets for GSM7498436

Status

Public on Nov 29, 2023

Title

HbhA_KO_Sauton_rep3

Sample type

SRA

Source name

H37Rv

Organism

Mycobacterium tuberculosis

Characteristics

strain: H37Rv
genotype: HbhA KO
treatment: Sauton

Treatment protocol

Mto3∆hbhA was inoculated at 0.05 OD, in 7H9 and Sauton's media. At 0.8 OD total RNA was islolated and RNA seq was performed.

Growth protocol

hbhA mutant strains was grown in 7H9 + ADC and Sauton's media till 0.8 OD. Mycobacterial strains was cultured in Middlebrook 7H9 medium (BD Biosciences) supplemented with 10% albumin, dextrose, NaCl, catalase (ADC) along with 0.2% glycerol (Sigma) and 0.05% Tween-80 (Sigma).

Extracted molecule

total RNA

Extraction protocol

RNA was isolated from exponentially growing cultures equivalent to 10 O.D.600units. The collected cells were resuspended in TRIzol reagent (Invitrogen), lysed using a bead beater (MP FastPrep system, MP Biomedicals) as per manufacturer's instructions, and the TRIzol-lysed cell mix was extracted with chloroform, followed by precipitation of RNA using isopropanol. The RNA pellet was dissolved in RNase-free water (30µl with 2µl of RiboLock (Thermo Scientific). DNA contamination was removed by DNase I (Invitrogen) treatment, and the RNA was further purified using RNeasy mini columns (Qiagen).
Total RNA was quantified using Qubit RNA BR kit and quality was assessed using Agilent 4200 Tapestation. Libraries were generated using Illumina stranded total RNA ribozero plus.
Final library was quantified using Qubit ds DNA HS kit and sequenced on Illumina NovaSeq 6000 on SP flow cell.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NovaSeq 6000

Data processing

Illumina adapters and low-quality reads were removed from raw sequencing reads using cutadapt. Reads with quality scores less than 20 and smaller than 36 bp were discarded.
The processed reads were then mapped to the Mycobacterium tuberculosis H37RV, downloaded from https://ftp.ncbi.nlm.nih.gov/genomes/refseq/bacteria/Mycobacterium_tuberculosis/reference/GCF_000195955.2_ASM19595v2/, using hisat2 with default parameters.
Uniquely aligned reads were counted using featureCounts of Subread package. There were 4008 genes in the gtf file, downloaded from https://ftp.ncbi.nlm.nih.gov/genomes/refseq/bacteria/Mycobacterium_tuberculosis/reference/GCF_000195955.2_ASM19595v2/, for which we had count information. Genes with total read count 10 across all the samples were removed resulting in 3987 genes for SigA knockdown experiment and 3999 genes for SigB knockout experiment, which were used for further analysis.
Differential gene expression analysis was performed using DESeq2. Genes with adjusted p-value < 0.05 and absolute log2 Fold change > 0.5 were considered differentially expressed.
For PCA plot and heat map, the raw read counts were rlog normalized, available with the DESeq2 package.
Assembly: Mycobacterium tuberculosis H37RV GCF_000195955.2_ASM19595v2
Supplementary files format and content: Count file in csv format that has uniquely aligned reads mapped to each gene, counted using featureCounts.

Submission date

Jun 20, 2023

Last update date

Nov 29, 2023

Contact name

Nitesh Kumar Singh

E-mail(s)

[email protected]

Organization name

Center for Cellular and Molecular Biology

Department

Bioinformatics