Ge sample loading reagent

Total RNA was extracted from PBMCs using the QIAamp RNA Mini Kit following the manufacturer’s instructions. The random primer was used to synthesis cDNA following the standard protocol of SuperScript III RT kit (Invitrogen). The synthesized cDNA was added to 1.5ml tube added with 190ul MILIQ H2O. The diluted cDNA was added with 200ul saturated phenolic chloroform mixture in the ice for 10min and centrifuged at 12000g for 10min. The upper aqueous phase was collected into a new 1.5ml tube added with 2ug glycogen and 500ul ethanol. After stored at -80 degree refrigerator for 8h, the sample was centrifuged at 14000g for 30min. The pellet was wash with 1ml 70% ethanol and centrifuged at 10000g for 5min. The cDNA was dissolved in 10ul H2O. cDNA from PBMCs was screened for expression of housekeeping genes ACTB and GAPDH and then were analyzed with TaqMan Universal PCR Master Mix (Applied Biosystems, Foster City, CA, USA), 20X GE Sample Loading Reagent (Fluidigm, South San Francisco, CA, USA), 2X Assay Loading Reagent (Fluidigm, South San Francisco, CA, USA) and individual TaqMan Gene Expression Assay using 96.96 Dynamic Array™ IFC (Fluidigm, South San Francisco, CA, USA) on a BioMark HD System (Fluidigm, South San Francisco, CA, USA). Ct (threshold cycle) values were processed and analyzed using the BioMark Real-Time PCR Analysis software (Fluidigm South San Francisco, CA, USA).

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.

PCR was performed following Gene Expression Standard TaqMan Assays protocol (Fluidigm cat n° 100–6170 C1), using a 10X assays mix and a pre-sample mix prepared separately. The 10x assays mix was prepared by mixing 2 μL of combined Primer (Forward/Reverse 6.7uM, Probe 1.7 μM) and 2ul 2X Assay Loading Reagent (Fluidigm PN 100–7611) to a final volume of 4 μL (per reaction). The pre-sample mix was prepared by mixing 2 μL TaqMan Universal PCR Master Mix (2X) (Life Technologies PN 4304437) and 0.2 μL 20X GE Sample Loading Reagent (Fluidigm PN 100–7610) and 1.8 μL preamplified cDNA to a final volume of 4 μL.
Then, 3 μL of 10x assays mix and of pre-sample mix are transferred into the 192.24 IFC, loaded into the Biomark^TM IFC controller RX and transferred to the Biomark^TM HD apparatus. Thermal cycling conditions were as follows: 50°C for 120 s, 95°C for 600 s followed by 20 cycles of 95°C for 15 s, 60°C for 1min.

We proceeded as described in Valente et al. (2019) (link). Cells were sorted in RT-STA reaction mix from the CellsDirect One-Step qRT-PCR Kit (Life Technologies), reverse transcribed and specific target pre-amplified (20 cycles), according to the manufacturer’s procedures. Pre-amplified samples were diluted 5x with low EDTA TE buffer prior to qPCR analysis using 48.48 Dynamic Array IFCs and the BioMark TM HD System (Fluidigm). The same TaqMan gene expression assays (20x, Life Technologies) were individually diluted 1:1 with 2x assay loading reagent (Fluidigm). Pre-amplified samples were combined with TaqMan Universal Master Mix (Life Technologies) and 20x GE sample loading reagent (Fluidigm). Loading of the 48.48 Dynamic Array TM IFCs and qPCR cycling conditions followed the Fluidigm procedure for TaqMan gene expression assays.

The pre-amplified, ExoI-treated cDNA was diluted 1:5 prior to analysis through quantitative PCR (qPCR) with the microfluidic 96.96 Dynamic array Standard BioTools UK Ltd., London, UK) performed on a BioMark HD instrument (BioMark) (as described in [16 (link)]). Assay mixes were prepared by mixing 2.25 µL of 2X Assay Loading Reagent (Fluidigm), 2.5 µL of primer pair mix (1.15 µM) and 0.25 µL of low-EDTA TE buffer. Sample mixes were prepared by mixing 2.5 µL of TaqMan Gene Expression Master Mix (TFS), 0.25 µL of 20X EvaGreen DNA binding dye (Biotum), 0.25 µL of 20X GE Sample Loading reagent (Fluidigm) and 2 µL of pre-amplified, ExoI-treated cDNA (diluted 1:5). The thermal cycling conditions for qPCR were: thermal mix at 50 °C for 2 min, 70 °C for 30 min and 25 °C for 10 min, followed by a hot start step of 50 °C for 2 min, 95 °C for 10 min and then 30 cycles of 95 °C for 15 s and 60 °C for 60 s, with the fluorescence emission recorded after each cycling step. After the completion of the run, a melting curve of the amplified product was determined (60 °C for 3 s to 95 °C). Raw quantitation cycle (Cq) data were collated with the Real-Time PCR Analysis software v 3.1.3 (Fluidigm), setting the parameters of the quality threshold (0.65), baseline correction (derivative) and Cq threshold method to auto (global).