Transcriptional profiling of pax2a-Low and pax2a-High thyroid follicular cells in zebrafish

Thyroid follicular cells (TFCs) are responsible for generation, storage and release of thyroid hormone. Single-cell analysis of zebrafish thyroid gland demonstrated transcriptional heterogeneity within the TFC population (Gillotay et al., bioRxiv, 2020. doi: 10.1101/2020.01.13.891630. GEO dataset for single-cell RNA-Seq.: GSE133466). Particularly, TFC displayed transcriptional heterogeneity in the expression of pax2a, a transcription factor involved in differentiation and maturation of TFCs. To validate the genetic heterogeneity, we generated a pax2a knock-in line, in which mKO2 expression is driven by endogenous pax2a locus. Using Tg(tg:nls-EGFP); pax2a:mKO2-Knock-in, we sorted for TFCs (GFP+) and separated the pax2a-Low (mKO2-Low) and pax2a-High (mKO2-High) populations for NGS. Overall design: We used fluorescence-activated cell sorting (FACS) coupled with next generation RNA-Sequencing to profile pax2a-high and pax2a-low TFCs from 5 mpf animals. Cells were sorted directly into lysis buffer provided in ReliaPrep™ RNA Miniprep Systems (Promega Z6011) and mRNA isolated according to the manufactor's protocol. cDNA was generated using Ribozome depletion protocol followed by adapter ligation. Sequencing was performed on llumina NextSeq500. Reads were splice-aligned to the zebrafish genome, GRCz11, using HISAT2. Featurecounts was used to assign reads to exons thus eventually getting counts per gene.