Single-Cell Real-Time PCR Protocol

The cDNA template for real-time PCR was prepared as previously reported (14 (link)) from FeSP neurons isolated and purified as described above. In brief, total RNA was purified from 50 neurons per sample using Agencourt AMPure XP beads (Beckman Coulter, Brea, CA, USA) and reverse transcribed using SuperScript III reverse transcriptase (Thermo Fisher Scientific, Waltham, MA, USA) and the oligo(dT) primer (5′-TATAGAATTCGCGGCCGCTCGCGATAATACGACTCACTATAGGGCG(T)₂₄-3′). After the addition of a poly(A) tail with terminal deoxynucleotidyl transferase (Roche Diagnostics, Basel, Switzerland), second-strand cDNA was synthesized using MightyAmp DNA polymerase (Takara Bio, Shiga, Japan) and the tagging primer (5′-TATAGAATTCGCGGCCGCTCGCGA(T)₂₄-3′). The cDNA was then amplified by 18 cycles of suppression PCR using MightyAmp DNA polymerase (Takara Bio) and the 5′-aminated primer (5′-NH₂-GTATAGAATTCGCGGCCGCTCGCGAT-3′).
Real-time PCR was performed on the LightCycler 480 System II using the LightCycler 480 SYBR Green I Master (Roche Diagnostics). Melt curve analysis was performed to verify that a single amplicon was obtained in each sample. Normalization was performed using the geometric mean of actb (GenBank accession number: NM_001104808) and eef1a (GenBank accession number: NM_001104662), according to Vandesompele et al. (47 ). The primers used for real-time PCR are listed in Table S3.