C1 single cell preamp

Single cells were captured and the loci of interest preamplified using the C1 system (Fluidigm, software version 2.2.1) following the manufacturer's protocol. Cells were partitioned using a 10–17 μm C1 Single-Cell Preamp integrated fluidic circuit (IFC) (PN 100-5479; Fluidigm). IFC priming, cell loading and lysis, reverse transcription, and preamplification were then carried out using reagents from the following kits: C1 Single-Cell Auto Prep Reagent Kit (PN 100-5139; Fluidigm), Ambion Single Cell-to-CT qRT-PCR Kit (4458237; Thermo Fisher Scientific), and 20X TaqMan Gene Expression primers (Thermo Fisher Scientific). Amplicons were transferred from the IFC to a 96-well plate and stored at −20°C.

The C1 Single-Cell Auto Prep System (Fluidigm, South San Francisco, CA, USA) was used for single-cell isolation and pre-amplification to prepare separate single-cell cDNA in a 5–10 µm C1 Single-Cell PreAmp Integrated Fluidic Circuit (IFC) within a C1-Chip. For single-cell isolation, a cell suspension of at least 660,000 cells/ml was used, which enabled at least 2000 cells to enter the C1-chip. Visualization of cell loading (empty, single, doublets, or debris) was done using a light microscope. Single-cell capture rates were documented. Cell lysis, reverse transcription, and pre-amplification were performed on the C1-chip. cDNA of each cell was collected for qRT-PCR preparation. cDNAs and 48 TaqMan gene expression assays (Thermo Fisher Scientific), including B2M (beta-2-microglobulin) and an RNA spike-in (spike 1) as control values, were applied to the BioMark Gene Expression 48.48 IFC for gene expression analysis. Information on all TaqMan gene expression assays used are listed in Supplementary Table 2. Detailed workflow can be found in the Fluidigm Real-Time PCR User Guide (PN 68000088).