RNA-Sequencing of Wild Type and lola-O mutant embryos. Drosophila melanogaster

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: 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.

纵向缺失基因（Longitudinals lacking，简称lola）是黑腹果蝇（Drosophila melanogaster）中最为复杂的基因之一，可编码多达20种不同的蛋白质异构体，并在轴突导向与神经重编程过程中作为关键转录因子发挥功能。
为更全面地表征Lola的功能，我们利用CRISPR/Cas9系统针对每个蛋白质异构体构建了特异性突变体。本靶向筛选实验不仅使我们能够重新审视此前已被证实的部分异构体功能，还可将已知功能分配至特定异构体，并揭示了特定变体在章鱼胺能通路中的关键作用。综上，本综合性研究拓展了Lola功能的已知谱系，同时证实CRISPR/Cas9系统是系统性解析体内复杂基因座功能的有效工具。
实验设计：将胚胎于25℃条件下收集2小时，随后继续发育20小时。之后将胚胎转移至TRIzol试剂（Thermo Fisher Scientific）中，按照制造商提供的操作流程提取总RNA。使用脱氧核糖核酸酶I（DNase I，NEB）按照厂商提供的方案处理RNA，随后采用NEBNext® Ultra™ 定向RNA文库制备试剂盒（适配Illumina®平台）完成文库构建。本实验以1 µg总RNA作为文库制备的起始原料。