C1 suspension reagent

A cell suspension obtained after FACS sorting was concentrated at a range of 600-1000 cells/μL. C1 Suspension Reagent was added (all ‘C1’ reagents were from Fluidigm, Inc.) in a ratio of 4 μL to every 7 μL cell suspension as previously described (Zeisel et al., 2015 (link)). 11 μL of the cell suspension mix was loaded on a C1 Single-Cell AutoPrep IFC microfluidic chip designed for 10- to 17-μm cells, and the chip was then processed on a Fluidigm C1 instrument using the ‘mRNA Seq: Cell Load (1772x/1773x)’ script (30 min at 4°C). The plate was then transferred to an automated microscope (Nikon TE2000E), where a brightfield and RFP or EGFP fluorescence image (20 × magnification) was acquired for each capture site using μManager (http://micro-manager.org/ (2)), which took < 15 min. Quality of cells and control for doublets was performed after each experiment as described in (Zeisel et al., 2015 (link)).

Cell suspensions in a concentration of 600-1000 cells/μL was used. C1 Suspension Reagent was added (all 'C1' reagents were from Fluidigm, Inc.) in a ratio of 4μL to every 7μL cell suspension. 11μL of the cell suspension mix was loaded on a C1 Single-Cell AutoPrep IFC microfluidic chip designed for 10- to 17μm cells, and the chip was then processed on a Fluidigm C1 instrument using the 'mRNA Seq: Cell Load (1772x/1773x)' script (30 min at 4°C). The plate was then transferred to an automated microscope (Nikon TE2000E), and a bright-field image (20× magnification) was acquired for each capture site using μManager (http://micro-manager.org/ (2)), which took <15 minutes. Quality of cells, control for doublets and processing of C1 chips were performed in the Eukaryotic Single Cell Genomics facility at SciLife Lab, as described in Zeisel et al. (2015) (link) and Marques et al. (2016) (link).