C1tm system

Single cell capture, specific reverse transcription of miRNAs, and cDNA pre-amplification were performed using the Fluidigm^® C1^TM System. The cells were loaded in the C1^TM Single-Cell Preamp IFC, for cell size 10–17 μm (Fluidigm) according to the manufacturer’s protocol (PN 100-6667). Pre-amplification was performed using Megaplex PreAmp Pool A primers (Thermo Scientific) and Single Cell PreAmp Mix (Ambion). Cells were stained with a LIVE/DEAD fluorescent assay (Thermo Scientific) to identify presence of living cells. All cell capture sites were manually inspected on an EVOS FL fluorescent microscope (Thermo Scientific); capture sites containing debris, multiple or dead cells, or no cells were excluded from further analysis (Figure 2C and Table 2).

F10 nutrient mixture (HAM), Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), horse serum (HS), penicillin/streptomycin (P/S), and Fungizone antimycotic (FZ) were obtained from Invitrogen (Taastrup, Denmark). CD56-conjugated microbeads were from Miltenyi Biotec (Lund, Sweden). C1^TM System and C1 integrated fluidic circuits (IFCs) were from Fluidigm (San Francisco, CA, United States). LIVE/DEAD cell staining solution, ActinRed and NucBlue ReadyProbes antibodies, and Megaplex PreAmp primers were from Thermo Scientific (Taastrup, Denmark).