Ion Torrent Proton sequencing; RNA-seq of Human Tuberculosis (TB) in active TB patients, contacts with latent infection and uninfected contacts
RNA-seq of Human Tuberculosis (TB) in active TB patients, contacts with latent infection and uninfected contacts
Whole blood RNA was isolated from 2 ml of blood collected in EDTA-coated vacutainer tubes. Plasma was removed and erythrocytes lysed before RNA isolation.  RNA was isolated using the  QIAamp RNA Blood Mini kit for on-column extraction following the manufacturer´s instructions.  Poly(A)-mRNA fraction was enriched with the Dynabeads® mRNA DIRECT™ Micro Kit (Thermo Fisher Scientific; CA, USA) according to the manufacturer's protocol. Enriched mRNA was used to prepare barcoded libraries with the Ion Total RNA-Seq Kit v2 (Life technologies- Thermo Fisher Scientific; CA, USA). Briefly, Poly(A) mRNA was fragmented with RNase III and purified using magnetic Nucleic Acid Binding Beads. Samples with an average size of 100–200 nucleotides were used for the construction of the whole transcriptome library. Unique 5´ and 3´ adapters were used to preserve the directionality of the strand. Fragments were retro-transcribed to cDNA. cDNA was amplified and used for template preparation. Each library template was clonally amplified on Ion Sphere particles (emulsion PCR).
Experimental Factor: disease
tuberculosis
Experimental Factor: disease staging
latent infection