Single-cell transcriptome profiling of zebrafish pancreatic beta-cells segregated by CaMPARI-based calcium labeling.
Coordination of cell activity trough Ca2+ waves enables pancreatic ß-cells to secrete precise quantities of insulin in response to blood sugar. However, how the coordinating Ca2+ waves are orchestrated in space and time remains unknown. By applying functional Ca2+ imaging  using CaMPARI and single-molecule RNA detection, we reveal a gene-signature depicting a molecularly and metabolically distinct state of first responder ß-cells.  We highlight Vitamin B6 metabolism as a signature pathway associated with the first responder ß-cells . Overall design: Ex vivo photoconversion of ins:CaMPARI islets and Fluorescence-activated cell sorting (FACS) The primary islets from 4 months post-fertilization Tg(ins:CaMPARI) animals were dissected from the fish after euthanasia.  Each islet was placed in a well of 24-well plate with 5 ml of DPBS medium supplemented with calcium, magnesium (ThermoFisher, 14040141), along with the appropriate concentration of D-Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently, the entire 24-well plate was photoconverted using a 405 nm LED array (Loctite, 1167593) for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller (Loctite, 1359255) was utilized for precise timing of light exposure.  The photoconverted islets were dissociated into single cells by incubation in TrypLE (ThermoFisher, 12563029) at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation, TrypLE was inactivated with 10% FBS, and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded, and the pellet re-suspended in 500 µl of HBSS (without Ca2+, Mg2+).  To remove debris, the solution was passed over a 30 µm cell filter (Miltenyi Biotec, 130-041-407).  To remove dead cells, calcein violet (ThermoFisher, C34858) was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC (Fluorescein isothiocyanate) for green fluorescence; and PE (Phycoerythrin) for red fluorescence.  Single-cell RNA-Sequencing For single-cell RNA-Seq, ß-cells isolated from Tg(ins:CaMPARI) islets were subjected to profiling using Smart-Seq2 protocol from Takara.  For this, islets were isolated, photoconverted ex vivo, dissociated and FAC-sorted as described above.  ß-cells were collected from two conditions: responsive ß-cells displaying photoconversion at 2.5 mM; and non-responsive ß-cells without photoconversion at 16.5 mM.  Photoconversion was defined as a high red-to-green ratio.  For FACS of each condition, index sorting was utilized, and the cells were collected in 96-well plate containing lysis buffer (2 µl of nuclease free water with 0.2% Triton-X 100 and 4 U murine RNase Inhibitor (NEB)).  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication (Singh et al., Scientific Reports, 2018; doi: 10.1038/s41598-018-35218-5).