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RNA-seq analysis of 2851 Knockdown with and without ATC

ABSTRACT: RNA-seq analysis of 2851 Knockdown with and without ATC

PROVIDER: PRJNA1269056 | ENA |

REPOSITORIES: ENA

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RNA-seq analysis of 2851 Knockdown with and without ATC

Project description:We have performred RNA-seq analysis of 2851 Knockdown to investigate the role of this gene in maintaning the homoeostasis of bacterium. RNA was extracted from exponentially growing mycobacterial cells in Middlebrook 7H9 media. For cells with ATC and without ATC, re-suspended in media , and finally the cells were grown further for 0.4-0.6OD at 37°C. After incubation cells were 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). RNA integrity was checked using Agilent 2200 Tape Station system (Agilent Technologies). Library construction, RNA-sequencing and data analysis have been carried out by Medgenome Labs Private Limited , India. The main purpose of this study is to understand how mycobacteria can sense acidic stress conditions and mount an appropriate stress response.

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