Transcriptomic analysis of Wild type M.tb H37Rv, HigB KO M.tb H37Rv and complemented Hig B KO M.tb H37Rv
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PROVIDER: PRJNA743563 | ENA |
REPOSITORIES: ENA
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Project description:Gene expression microarray was performed using early log phase culture at 37 degree and 200 rpm followed by RNA extraction from M.tb H37Rv, HigB KO M.tb H37Rv and complemented Hig B KO M.tb H37Rv For gene expression profiling, total RNA was isolated from wild type H37Rv, higB KO mutant strain and complemented strains. The bacterial pellets for M.tb H37Rv wild type, M.tb H37Rv HigB KO mutant strain and complemented strain were resuspended in 1.0 ml TRIzol (Invitrogen Corporation, Carlsbad, CA); mRNA was extracted as per standard protocols and cleaned using RNAeasy columns (Qiagen GmbH, Hilden, Germany). Extracted mRNA (1 μg) was treated with DNase I using the Ambion DNA-free kit (Invitrogen Corporation, Carlsbad, CA) to remove genomic DNA contamination and quantified using Nanodrop 2000c spectrophotometer (Thermo Scientific, USA). The purity and integrity of RNA samples were assessed Agilent 2100 Bio analyzer (Agilent TechnologiesInc., USA). Further, 25ng of RNA was amplified and labeled using Low input Quick Amp WT Labeling kit (Agilent Technologies, USA). The labeled cRNA was purified using RNAeasy columns (Qiagen, USA) and total yields were quantified using Nanodrop 2000c spectrophotometer.
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Project description:To explore the regulatory network of noncoding RNAs after M.tb infection and the role of Rv1759c in the infection process, we collected samples of H37Rv- and H37Rv△1759c-infected macrophages and explored the full transcriptome expression profile. We constructed DE-lncRNA/DE-circRNA-DE-miRNA-DE-mRNA regulatory networks during H37Rv and H37Rv△1759c infection. In addition, we first discovered the close relationship between Rv1759c and chemokines during M.tb infection by comparing the transcription profiles of H37Rv and H37Rv△1759c and bioinformatics analysis. Here, our study comprehensively characterizes the ncRNA and mRNA profiles in macrophages infected with H37Rv and H37Rv△1759c, which provides support and new directions for in-depth exploration of ncRNA and PE/PPE family functions during the infection process.
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Project description:Transcriptional profiling of M.tb H37Rv cells comparing control wild type H37Rv with H37Rv cells electroporated with constitutive expression plasmid pVV16 expressing ESAT-6 binding peptide SL3. The expression of SL3 makes H37Rv less virulent during ex vivo and BalB/c mice infections, sequesters ESAT-6 inside M.tb cells and cause severe defects in mycobacterial morphology. Goal was to determine the effects of SL3 expression on global H37Rv gene expression. Two color Experiment,Organism: Mycobacterium Tuberculosis, ilife Discoveries designed Custom Mycobacterium tuberculosis on 8x15k GE Microarray. Two-condition experiment, H37Rv vs. H37Rv/SL3. Biological replicates: 2 biological control H37RV replicates labelled with Cy3, 2 SL3 biological expressing replicates labelled with Cy5.
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Project description:In this study, RNA sequencing (Transcriptome sequencing) was employed to investigate the global transcriptome changes in the macrophages during the different strains of M.tb infection. THP-1 cells derived macrophages were exposed to the virulent M.tb strain H37Rv (Rv) or the avirulent M.tb strain H37Ra (Ra), and the M.tb BCG vaccine strain was used as a control. The cDNA libraries were prepared from M.tb infected macrophages and then sequenced.
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