NextSeq 500 paired end sequencing; Monocytes from people with active or latent TB stimulated in vitro with PPD
CD14+ monocytes were isolated from peripheral blood mononuclear cells (PBMC) using magnetic positive selection (Miltenyi Biotec). Monocyte culture lysates were collected in RLT buffer (Qiagen) and total RNA purified using the RNeasy Mini kit (Qiagen). RNA was subjected to DNase treatment to remove contaminating genomic DNA using a TURBO DNA-free kit (Ambion, Life Technologies). Concentration and quality of all RNA samples were assessed using the Nanodrop Spectrophotometer and Agilent Bioanalyzer, respectively. The KAPA mRNA HyperPrep Kit (Roche Diagnostics) was used to construct stranded mRNA-Seq libraries from up to 500 ng intact total RNA.