Single-nucleus RNA sequencing of the developing wings of ivory Delta78k mutants in Heliconius melpomene butterflies

CRISPR studies show that the ivory:mir-193 gene is involved in the specification of color scale types in Heliconius butterflies. In order to examine the transcriptional effects of mir-193 loss-of-function on wing pattern development, we used the previously described Piano Keys line of Heliconius melpomene, which carries a recessive, 78-kb deletion that removes the 3' portion of the ivory lncRNA and the whole of miR-193. Homozygous mutant Delta78k butterflies phenocopy the most extensive knockout phenotypes of ivory:mir-193, while heterozygotes retain malformed red scales. Pupal wings were dissected, snap-frozen, and genotyped by PCR, allowing the selection of individuals that were Delta78k -/-, -/+, or +/+ for single-nucleus RNA-seq analysis (snRNA-seq). We selected one pupa at 30% of pupal development with homozygous wild-type ivory:miR193, one heterozygous Delta78k pupa at 30-32% pupal development, and three Delta78k homozygotes at 30-32%, 40-42%, and 50-52%