RNA-Sequencing of Wild Type, Lola-F and Lola-Null 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 13 hours. lola-F and lola-null embryos were hand-sorted and collected based on the expression of mCherry. Control embryos were collected in parallel and both mutant and control 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 manufacturers 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.