Bulk RNA-seq of fin and body melanocytes and microenvironments. Bulk RNA-seq of fin and body melanocytes and microenvironments

Broadly we were interested in understanding how fin and body melanocytes were different. The goal of the experiment was to investigate the interaction between three different variables: (1) cell lineage: melanocyte vs bulk microenvironment, (2) anatomic location: fin vs body, (3) genotype: wildtype vs CRKL,GAB2,TERT overexpression with NF1 loss. We found that melanocytes have unique transcriptional programs based on their anatomic location and that these programs can regulate the response to oncogenic drivers like CRKL. Overall design: For this experiment we used two genetic strains of zebrafish. (1) Wildtype melanocyte model stable line was generated by injecting Caspers with MiniCoopR-eGFP. (2) Acral melanoma model stable line was generated by injecting Caspers with MiniCoopR-eGFP, mitfa:hsCRKL, mitfa:hsGAB2, mitfa:hsTERT, mitfa:Cas9-mCherry;U6-nf1a-gRNA, mitfa:Cas9-mCherry;U6-nf1b-gRNA. Fish were dissected to collect body skin and fins, digested using liberase, and then FACS sorted for GFP+ melanocytes and GFP- microenvironment cells. Each biological replicate constituted a pooling of 2 males and 2 females. There were 2 genetic strains(WT_v_Acral) x 2 anatomic locations(fin_v_body) x 2 sorted conditions(GFP+_v_GFP-) x 3 biological replicates = 24 samples total.