Superscript 3 platinum taq

Amplification reactions were done using the CellsDirect kit (Thermo-Fisher) essentially according to manufacturer’s instructions, with the following modifications. The 31 gene multiplex primer set was added to individual lysates (100 nM final) in a final volume of 10 μL. Tubes were heated at 80°C for 10 min and chilled on ice for 3 min. 10 μL of 2x reaction buffer and 1 μL of SuperScript III/Platinum Taq (Thermo-Fisher) were added and tubes were reacted in a PCR machine at 50°C for one hour, followed by 85°C for 15 min to inactivate the reverse transcriptase. PCR amplification was then performed with an initial activation at 94°C for 2 min, followed by 18 cycles of 94°C for 30 s, 60°C for 30 s, and 72°C for 30 s. After amplification, 20 μL of 10 mM Tris 7.5 was added to each sample to bring up the total volume to 40 μL. Four μL of each sample was then screened by quantitative PCR to determine expression levels of Gapdh (indicating successful capture and amplification) and Ncam1 (indicating an OSN). Taqman primers were designed to amplify regions internal to the 31 gene multiplex primer sequence, and samples were run on an ABI 7500 (Thermo-Fisher). Only cells with Ct values ≤ 25 for both genes were used for the NanoString analysis (Seattle, WA). See Figure 1—source data 1.

OpenArrays were custom designed by ThermoFisher with the Taqman assay ID shown in Appendix Table S4. Single cells were directly deposited by Fluorescence Activated Cell Sorting into 9 μl of a pre‐amplification mixture (CellDirect One‐Step qRT‐PCR kit, 11753‐500) which contains 0.05× of each TaqMan assay, 1× CellDirect reaction mix, 200 ng/μl SuperscriptIII/Platinum Taq and 100 ng/μl SUPERase‐In (ThermoFisher) in DNA suspension buffer (TEKnova). The reverse transcription and gene‐specific PCR amplification was carried out in a thermal cycler with the following condition: 50°C for 30 min, 95°C for 2 min followed by 24 cycles of 95°C for 15 s, 60°C for 4 min. cDNA was diluted 1:10, and only cells with at least two housekeeping genes amplified were chosen for whole panel gene expression profiling. The cDNA samples were loaded onto an OpenArray using OpenArray AccuFill system, and the quantitative real‐time PCR was run using Quantstudio 12K Flex System. For gene expression analysis, the average of five housekeeping genes (Actβ, Gapdh, Tbp, Ppia, Atp5a1) was used for normalisation.
For quality control, the expression of Actβ and Atp5a1 was first analysed to establish whether the cells were deposited successfully. We excluded wells where no amplification or abnormal amplification was obtained.