Single-cell transcriptomics of Drosophila optic lobe

We crossed the Gal4 lines specific for individual cell types (Pm2-Gal4 (Bl stock no. 50240), T4-Gal4 (VT stock no. 37588)) as well as pan-neuronal marker line (Elav-Gal4) and pan-glial marker line (Repo-Gal4) to UAS-Red Stinger to fluorescently label nuclei of individual cell types, sort and sequence them as previously described^{18 (link)}. Briefly, we first dissected adult optic lobes, then dissociated tissue while maintaining cell viability and finally, sorted cells by FACS (Facs Aria III) based on their differences in fluorescence intensity and cell size. RNA was extracted using the Arcturus PicoPure RNA Isolation Kit (Applied Biosystems) and the Smart-Seq v4 Ultra Low Input RNA Kit (Clontech) was used to generate full-length double stranded cDNA with 300 to 500 pg of total RNA input. The quality of RNA and cDNA was assessed by Bioanalyzer using RNA 6000 Pico and High sensitivity DNA assay (Agilent) respectively. Libraries were prepared using Nextera XT DNA Library Prep Kit (Illumina) and run on the Illumina HiSeq 2500 (CGSB at NYUAD). Three barcoded libraries were pooled per sequencing lane and paired-end 100 bp reads were generated. Sequences were mapped to the Drosophila melanogaster genome (BDGP6.81) using TopHat2 (v2.1.0). We obtained three biological replicates for each library.