High-throughput Gene Expression Analysis

We selected a total of 87 genes based on broad relevance to inflammation, immunopathology, antimycobacterial response, type I IFN response, immune regulation as well as risk for TB (see Table S1 in Supplementary Material). Expression levels of these genes were measured using the previously described high-throughput microfluidic 96.96 Dynamic Array and BioMark™ HD instrument (Fluidigm, USA) (24 (link)). Specifically, a 5 µl sample mix was prepared for each sample containing 2.5 µl of 2X TaqMan Universal PCR Master Mix (Applied Biosystems, PN 4304437), 0.25 µl of 20X GE Sample Loading Reagent (Fluidigm PN 85000746) and 2.25 µl of pre-amplified cDNA (diluted 1:25). 5 µl of assay mix was prepared with 2.5 µl of each 20X TaqMan GE Assay (Thermo Fisher Scientific, USA) and 2.5 µl of 2X Assay Loading Reagent (Fluidigm PN 85000736). An IFC Controller HX (Fluidigm, USA) was used to prime the Dynamic Array™ (chip) with control line fluid and before loading the sample and assay mixes into their appropriate inlets. The chip was subsequently returned to the IFC Controller HX for loading and mixing. After approximately 60 min, the chip was transferred to the BioMark™ HD instrument for RT-qPCR according to the manufacturer’s protocol.