RNA-Sequencing of FACS sorted neurons and octopaminergic cells

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. Overall design: For the neuronal control, embryos were collected at 25°C for three hours and subsequently developed for 13 hours. For octopaminergic cells, embryos were collected for three hours and developed for further 20 hours. Cells were isolated as previously described (Salman and Perrin, 2011) and FACS sorted. Per replicate, 10 cells were collected in lysis buffer and subjected to library preparation using the Smart-Seq2 Kit for Illumina.