Single-cell RNA sequencing of zebrafish thyroid cells

The thyroid gland is responsible for supplying the thyroid hormones to the body. The gland is an endocrine organ with an intricate structure enabling production, storage and release of the thyroid hormones. The gland is composed of numerous spherical follicles of varying sizes, surrounded by thyroid follicular epithelial cells, or thyrocytes. The thyrocytes surrounding the follicles generate the thyroid hormones in a multi-step process. Though the machinery responsible for the production of thyroid hormones by thyrocytes is well established, it remains unknown if all the thyrocytes resident in the thyroid gland are equally capable of generating thyroid hormones. In other words, the extent of molecular homogeneity between individual thyrocytes has not yet been investigated. To obtain an unbiased picture into the molecular heterogeneity present in zebrafish thyrocytes, we performed droplet based next-generation sequencing of individual thyrod gland cells. Using unsupervised clustering, we could identify all the major cell types present in the thyroid gland. Moreover, we could define sub-populations within the major cell-types, demonstrating the presence of molecular heterogeneity within nominally homogenous cell-populations. We used 10x Genomics to profile thyroid cells from zebrafish. Thyroid gland was enzymatically 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 6000 (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.