RNA-Sequencing of Wild Type and lola-O mutant embryos

Longitudinals lacking (lola) is among the most complex genes in Drosophila melanogaster, encoding up to 20 different protein isoforms and acting as a key transcription factor in axonal pathfinding and neural reprograming. To better characterize Lola function we have generated specific mutations in each isoform using the CRISPR/Cas9 system. Our targeted screen allows us to revisit the previously demonstrated roles for few isoforms, to assign known functions to specific isoforms and to reveal a critical role for a specific variant in the octopaminergic pathway. Thus, our comprehensive study expands the repertoire of Lola functions, and demonstrates that the CRISPR/Cas9 approach is a valuable tool to systematically address the role of complex loci in vivo. Embryos were collected at 25°C for two hours and subsequently developed for 20 hours. Embryos were subsequently transferred into TRIzol reagent (Thermo Fisher Scientific) and RNA was isolated using the manufacturers protocol. RNA was DNase I (NEB) treated according to the manufacturer's protocol and subjected to library preparation using the NEBNext® Ultra™ Directional RNA Library Prep Kit for Illumina®. 1 µg of total RNA was used as starting material for library preparation.