Hx ifc controller

This was carried out using a Biomark HD system (Data Collection software version 4.1.3) and IFC Controller HX (Fluidigm) following the manufacturer’s quick reference guide. Gene expression analysis was carried out using Dynamic Array Flex Six Gene Expression IFCs (Fluidigm). Assays were performed using the Flex Six Gene Expression Reagent Kit (Fluidigm) as per manufacturer’s instructions with 20× TaqMan Gene Expression primers (Thermo Fisher Scientific) and TaqMan Fast Advanced Master Mix (Thermo Fisher Scientific). Cells (both treated and untreated) where analysed from three separate cultures. After quality control 115 cells were included in the final analysis (52 treated and 64 control).

Allele-specific SNPtype assays were run using the Fluidigm Flex Six™ Genotyping IFC (Fluidigm Corp., South San Francisco, CA, USA). Specific target amplification (STA) was performed to increase the number of molecular targets at the beginning. The determined thermal cycle program was run using the Bioer Gene Pro Thermal cycler (95°C for 15 min followed by 14 cycles of 95°C for 15 sec and 60°C for 4 min). SNPtype assay mixes and sample mixes were prepared according to the manufacturer’s protocol. After all the dynamic array was loaded with a 4 μL of each 10× assay mix and 5 μL of each sample mix. Dynamic array was placed on the IFC Controller HX (Fluidigm), and the loading process was completed. Then, dynamic array was placed on the BioMark system (Fluidigm), which performs the thermal cycling and fluorescent image acquisition. A data collection software was used on BioMark system. Genotyping application, ROX passive reference, and SNPtype-FAM and SNPtype-HEX probe types were selected. The SNPtype E FLEXsix v1 protocol was used for thermal cycling and image capture. At the end, we assessed the genotypes of the samples.

Glioblastoma tissues were obtained from the operating room and immediately dissociated using a papain-based dissociation system (Worthington Biochemical). Leukocytes were depleted using anti-human CD45 MicroBeads (Miltenyi Biotec). The C1 single-cell auto prep instrument (Fluidigm) was used for capturing single cells. Pre-amplified cDNA was utilized for qPCR with the Biomark HD system with IFC Controller HX (Fluidigm) and 2× SsoFAST EvaGreen supermix with low ROX (Bio-Rad).

Single SSEA1+ cells were sorted into 96 well plates containing 5 µl 2x CellsDirect reaction mix and stored at -80ºC until use. After thawing, the plates were processed according to the manufacturer’s instructions using 48 DELTAgene assays (Supplementary file 5). Each sorted 96 well plate was split in two identical parts to match loading onto 48.48 gene expression Dynamic Arrays (Fluidigm, South San Francisco, CA) and each 48 well section contained 14x single cells and 100 and 1000 cell controls for each cell line. Arrays were primed on an IFC Controller HX (48.48/digital; Fluidigm) and then scanned on a Biomark HD reader (Fluidigm). Fluidigm’s Real Time PCR Analysis software was used to assess melting curves and assays with multiple peaks were excluded. The remaining assays were then analyzed in R using the SINGuLAR Analysis Toolset 3.0.3 (Fluidigm). Outlier cells were removed from the analysis using the Outlier Analysis tool from this toolset. Technical information for single cell qPCR assays are outlined in Supplementary file 5.

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.

Microfluidic QPCR was performed on the 96.96 dynamic array (Fluidigm) using 2.25 µl of the diluted cDNA sample from the specific target amplification, along with Taqman assays for each gene, Taqman Universal Master Mix, and Fluidigm loading reagent as outlined in the manufacturer's protocol, by priming and loading the chip via the HX IFC Controller (Fluidigm) and performing the QPCR in the BioMark QPCR platform (Fluidigm), with default parameters for gene expression data collection as advised by Fluidigm.