Single-cell RNA sequencing of zebrafish pancreatic cells

The pancreatic beta-cells regulate blood glucose levels by secreting the hormone insulin in response to increasing glucose levels. Recent work has identified molecular and functional heterogeneity among the beta-cell community. To ontain an unbiased picture into the molecular heterogeneity present in zebrafish pancreatic cells, we performed droplet based next-generation sequencing of individual pancreatic cells. Using unsupervised clustering, we could identify all the major cell types present in the pancreas. Moreover, we could define sub-populations within the major cell-types, demonstrating the presence of molecular heterogeneity within nominally homogenous cell-populations. Overall design: We used 10x Genomics to profile pancreatic cells from zebrafish. Pancreatic islets from six animals were dissociated and single-cell library prepared using 10x Genomics Chromium pipeline. Sequencing was performed on llumina NextSeq 550 machine using a HighOutput flowcell in paired-end mode (R1: 26 cycles; I1: 8 cycles; R2: 57 cycles), thus generating ~45 mio fragments. The raw sequencing data was then processed with the 'count' command of the Cell Ranger software (v2.1.0) provided by 10X Genomics with the option '--expect-cells' set to 5000 (all other options were used as per default). To build the reference for Cell Ranger, zebrafish genome (GRCz10) as well as gene annotation (Ensembl 87) were downloaded from Ensembl and the annotation was filtered with the 'mkgtf' command of Cell Ranger (options: '--attribute=gene_biotype:protein_coding --attribute=gene_biotype:lincRNA –attribute=gene_biotype:antisense'). Genome sequence and filtered annotation were then used as input to the 'mkref' command of Cell Ranger to build the appropriate Cellranger Reference.