cDNA synthesis was performed using the Smart-seq2 protocol1. In brief, 384-well or 96-well plates containing single- cell lysates from previous step were thawed on ice followed by first-strand synthesis. 0.6 μl of reaction mix was added for 384-well plates and 6 μl of reaction mix to each well using the SPT LabTech DragonFly. All plates were sealed with Microseal Seal B adhesive seal and spun down (1000 rcf, 1 min). Reverse transcription was carried out by incubating wells on a ProFlex 2 × 384 thermal-cycler (Thermo Fisher) at 42 °C for 90 min, stopped by heating at 70 °C for 5 min and cooled to 4 °C hold. When completed, plates were either stored in −20 °C or immediately processed for 2nd strand synthesis and cDNA amplification.
SMARTScribe Reverse Transcriptase (Takara Bio, 639538)
Recombinant RNase Inhibitor (Takara Bio, 2313B)
5X First-Strand Buffer (Takara Bio, 639538),
100 μM TSO (Exiqon, 5′-AAGCAGTGGTATCAACGCAGAGTGAATrGrGrG-3′)
100 mM dithiothreitol (Bioworld, 40420001-1)
5 M Betaine (Sigma, B0300-5VL)
1 M MgCl2 (Sigma, M1028-10X1ML)
1. Picelli, S. et al. Smart-seq2 for sensitive full-length transcriptome profiling in single cells. Nat Methods 10, nmeth.2639 (2013).