cDNA library from single neurons were generated using Invitrogen SuperScript III CellsDirect cDNA Synthesiss Kit (ThermoFisher 18080300) and as previously described57 . In short, manually isolated neurons were collected into 0.2 mL thin-walled PCR tubes prefilled with 10 μl supplied lysis buffer and RNase inhibitor; and were flash frozen on dry ice and stored at -80°C until cDNA synthesis. DNase digestion was performed for all cells. First-strand cDNA was generated using 100 nmols (50 mM, 2μl) of oligo(dT)20. All other steps were performed according to the manufacturer’s protocol.
qPCR was performed using power SYBR Green master mix (ABI 4368702) on an ABI StepOnePlus qPCR machine. Single cell genomic DNA was used as the negative controls and FACS isolated DRG neurons (~36,000 neurons, diluted 1:36,000) was used as the positive control. Gapdh was used to identify and exclude samples without input or with failed cDNA synthesis. All primer sets were validated before use, and PCR products were selected for further sequence validation. All gene expression data is presented as folds of Gapdh expression, calculated using 2ˆ-(Ct(target Gene)-Ct(Gapdh)).
qPCR was performed using power SYBR Green master mix (ABI 4368702) on an ABI StepOnePlus qPCR machine. Single cell genomic DNA was used as the negative controls and FACS isolated DRG neurons (~36,000 neurons, diluted 1:36,000) was used as the positive control. Gapdh was used to identify and exclude samples without input or with failed cDNA synthesis. All primer sets were validated before use, and PCR products were selected for further sequence validation. All gene expression data is presented as folds of Gapdh expression, calculated using 2ˆ-(Ct(target Gene)-Ct(Gapdh)).