Illumina HiSeq 2500 paired end sequencing; The global transcriptome of whole lungs extracted from M. tuberculosis infected C57BL6 mice
The global transcriptome of whole lungs extracted from M. tuberculosis infected C57BL6 mice
Whole lungs were extracted, finely chopped, resuspended in Trizol. Mice were euthanized dissected following protocols approved by the competent authority as per Institutional guidelines. Mycobaterium tuberculosis were grown in 7H9 media supplemented with glycerol, tween and albumin-dextrose-catalase. Mice were grown following protocols approved by the competent authority as per Institutional guidelines. Mice were infected with 200 cfu of Mycobacterium tuberculosis per animal using a standard aerosolic chamber nebulizer. RNA was isolated using Illumina proprietary kit at a commercial facility. The mRNA of Eukaryote organisms is enriched from total RNA by Oligo (dT) beads and the RiboZero kit is used to remove rRNA from the Prokayote organisms. First, the mRNA is fragmented randomly by adding fragmentation buffer, then the cDNA is synthesized by using mRNA template and random hexamers primer, after which a custom second-strand synthesis buffer (Illumina) , dNTPs,RNase H and DNA polymerase I are added to initiate the second-strand synthesis. Second, after a series of terminal repair, a ligation and sequencing adaptor ligation, the double-stranded cDNA library is completed through size selection and PCR enrichment.