C1 preamp ifc

Cultured organoids were dissociated into single cells using TrypLE select (Thermo Fisher Scientific, Waltham, MA, USA) during the third culture passage. Single-cell suspensions were loaded onto the C1 preamp IFC (10–17 μm, Fluidigm San Francisco, CA, USA), for single-cell isolation. Single-cell capture was confirmed under a microscope (BX-X700, Keyence, Osaka, Japan). Pre-amplification of target genes was performed using TaqMan assays (Thermo Fisher Scientific, Waltham, MA, USA), with the IFC controller MX (Fluidigm, San Francisco, CA, USA). Further amplification and data acquirement of the multiplex quantitative PCR analysis was done using the Biomark 48.48. dynamic array IFC and Biomark HD system (Fluidigm, San Francisco, CA, USA), according to manufacturer’s instructions. The set of target genes, and the corresponding gene-specific TaqMan assays analyzed in the study can be found in the Suppl. Table S2.

Caps and rings were dissected from 3D-retinas. The cap whose ring passed the ring-PCR test was subjected to scPCR analysis. The cap was dissociated into single cells by using papain (Wako), followed by loaded into the C1 preamp IFC (10–17 μm, Fluidigm) for single cell isolation. Pre-amplification of genes was performed in the C1 instrument (Fluidigm) according to manufacturer’s instructions. The pre-amplified cDNAs from each cell were further amplified by using the Biomark HD instrument (Fluidigm) with the TaqMan assays.