C1 chip

GMM of islet marker genes was performed on a per gene basis using the R-package mclust_5.2 (Scrucca et al. 2016 (link)). Each single-cell sample was classified as a specific pancreatic cell type if and only if a single gene from the selected marker gene list—INS (beta), GCG (alpha), SST (delta), PPY (PP/gamma), KRT19 (ductal), PRSS1 (acinar), and COL1A1 (stellate)—was expressed in the sample and none of the other marker genes were expressed. Cells expressing no marker genes were labeled as “none,” and those expressing >1 marker gene were labeled as “multiple.” Fluidigm released a white paper report detailing the potential for single cells to “z-stack” in up to 30% of capture nests on the medium (10–17 µm) Fluidigm C1 chip (http://info.fluidigm.com/rs/673-MRG-416/images/C1-Med-96-IFC-Redesign_wp_101-3328B1_FINAL.pdf). DAPI staining identified z-stacked islet cell doublets in 10% and 30% of capture nests from two additional C1 single-cell captures. Because the proportion of “multiple” labeled cells approximately equaled that of z-stacked doublets, we discarded these cells (n = 340) from subsequent analyses.

Dissociated cells were FACS sorted based on fluorescence (RFP+ and EGFP+) either into a tube, prior to running them on the C1-STRT (Dataset A), or directly onto the STRT-seq-2i (Dataset B) platform. For Dataset A, BD FACSAria” III Cell Sorter B5/R3/V3 system was used to collect both fluorescent and non-fluorescent populations from Lhx6^cre::R26R-tdTomato and 5HT3a^EGFP mice, followed by a subsequent manual loading into the C1 chip (Fluidigm system). For the cortico-striatal comparison dataset cortical Pvalb^cre::TdT positive cells were sorted on a FACS ARIA II directly into 3 μL of ice cold ACSF-D with 0.5% BSA in the cell collection chamber of a Fluidigm C1 chip to a final concentration 100-150 cells/μL. The collected cells were processed immediately after FACS on the Fluidigm C1 System according to the C1-STRT protocol. For Dataset B Lhx6^cre::R26R-tdTomato and 5HT3a^EGFP positive cells only were sorted using BD FACSAria II SORP, straight onto the chip array (2400 cells/chip prefilled with 50 nL lysis buffer).