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Sample GSM7814647

Query DataSets for GSM7814647

Status

Public on Oct 02, 2023

Title

phoR KO 4.5-REPLICATE 3

Sample type

SRA

Source name

bacteria

Organism

Mycobacterium tuberculosis

Characteristics

strain: H37Rv
cell type: bacteria

Treatment protocol

Cultures were grown till mid log phase and then divided into two halves. One part was exposed to acidic stress (pH 4.5) and other one to normal conditions (pH 7.0) for 2 hours. RNA was isolated and processed for RNA sequencing.

Growth protocol

M. tuberculosis H37Rv strains were grown at 37°C in Middlebrook 7H9 broth (Difco) containing 0.2% glycerol, 0.05% Tween-80 and 10% Middlebrook ADC (albumin-dextrose-catalase).

Extracted molecule

total RNA

Extraction protocol

RNA was extracted from exponentially growing mycobacterial cells in Middlebrook 7H9 media. Briefly, 25 ml of bacterial culture was grown (in the indicated pH) to mid-log phase (OD600= 0.4 to 0.6) and combined with 40 ml of 5 M guanidinium thiocyanate solution containing 1% β-mercaptoethanol and 0.5% Tween 80. Cells were pelleted by centrifugation, and lysed by re-suspending in 1 ml Trizol (Ambion) in the presence of Lysing Matrix B (100 µm silica beads; MP Bio) using a FastPrep-24 bead beater (MP Bio) at a speed setting of 6.0 for 30 seconds. The procedure was repeated for 2-3 cycles with incubation on ice in between pulses. Next, cell lysates were centrifuged at 13000 rpm for 10 minutes; supernatant was collected and processed for RNA isolation using Direct-ZolTM RNA isolation kit (ZYMO) as per manufacturer’s recommendation. Following extraction, RNA was treated with DNAse I (Promega) to degrade contaminating DNA, and integrity was assessed using a Nanodrop (ND-1000, Spectrophotometer). RNA samples were further checked for intactness of 23S and 16S rRNA using formaldehyde-agarose gel electrophoresis, and Qubit fluoremeter (Invitrogen).
Total RNA was isolated using Direct-zol RNA MiniPrep kit(Zymo Research Corporation),thepurity of isolated RNA was determined using the NanoDrop (ThermoScientific), quantified using Qubit (Invitrogen), and RNA integrity was checked using Agilent 2200 Tape Station system (Agilent Technologies).Library construction, RNA-sequencing and data analysis have been carried out by Agrigenome Labs Private Limited (Cochin), India.Briefly,The rRNA was removed using ‘si-tools Pan-prokaryote ribopool probes,’ and further library preparation was carried out using TruSeq Stranded RNA library prep kit. Prepared libraries were sequenced using Illumina HiSeq x10 platform, and 150-bp paired-end reads were generated.Thereads that passed quality control were mapped onto the reference genome ofM. tuberculosisH37Rv usingHisat2(version-2.0.5).The abundance of transcripts in individual samples was estimated usingCufflinks (v2.2.1).

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2000

Data processing

Basecalling, demultiplication and adaptor trimming was performed by Illumina HiSeq control software
Quality of raw reads was assessed by using FastQC tool
Clean reads that passed the quality filter were mapped to the reference genome (Mycobacterium tuberculosis H37Rv accession number NC_000962.3) using Hisat2.
Differential gene expression analysis of samples with respect to control was performed using Cuffdiff program (v2.2.1).
Assembly: NC_000962.3
Supplementary files format and content: tab-delimited text files include FPKM values for each Sample ...

Submission date

Sep 29, 2023

Last update date

Oct 02, 2023

Contact name

Dibyendu Sarkar

E-mail(s)

dibyendu@imtech.res.in

Organization name

CSIR-IMTECH

Street address

SECTOR 39A