Computational protocol: Growth of Mycobacterium tuberculosis in vivo segregates with host macrophage metabolism and ontogeny

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Protocol publication

[…] RNA isolation and sequencing were done at the RNA Sequencing Core at Cornell University. Briefly, total RNA was extracted from FACS-purified lung macrophages using Trizol according to the commercial protocol, with the addition of a chloroform back-extraction step, addition of GlycoBlue (Thermo Fisher) as a carrier at the precipitation step, and a second 70% ethanol wash of the RNA pellet. All RNA samples (three biological replicates in each group) passed quality control analysis on a Fragment Analyzer (Advanced Analytical). Directional poly-A+ RNA-sequencing libraries were generated with the NEBNext Ultra II RNA library prep kit (New England BioLabs). Libraries were sequenced on a NextSeq500 (Illumina) for a mean of 41 million reads per sample. The Tophat program (version 2.1) was used to align the reads to the UCSC mm10 mouse reference genome, and the Cufflinks program (version 2.2) was used for the measurements of transcript abundance represented by fragments per kilobase of transcript per million mapped reads and statistical analysis of differential gene expression. RNA-sequencing data have been deposited under GEO accession no. GSE108844. […]

Pipeline specifications

Software tools TopHat, Cufflinks
Application RNA-seq analysis
Organisms Mycobacterium tuberculosis, Bacteria, Mus musculus
Diseases Infection, Tuberculosis
Chemicals Deoxyglucose