Single-Cell RNA Sequencing of Drosophila DH44+ Neurons

As described previously,^{37 (link)} individual DH44⁺ cells (visualized by GFP expression) were harvested from dissected fly brain under a fluorescence microscope with a glass micropipette pulled from thick-walled borosilicate capillaries (BF120-69-10, Sutter Instruments). Individual cells were immediately transferred to lysate buffer (0.9× PCR Reaction Buffer II, 1.35 mM MgCl₂, 0.45% NP40, 4.5 mM DTT, 0.18 U/µL SUPERase-In, 0.36 U/µL RNase inhibitor, 12.5 nM UP1 primer, 0.045 mM dNTP mix) and underwent reverse transcription and cDNA amplification (SMARTer Ultra Low RNA Kit for Sequencing, Clonetech).
To build the cDNA library, library preparation kit (NEBNext Ultra II DNA Library Prep Kit, NEB) was used. The amplified cDNAs gained from the former step were sonicated to ~250 bp fragments by the Covaris S2 system, and fragemented cDNAs were then subjected to end-preparation, adaptor ligation, adaptor-ligated DNA cleanup and PCR amplification successively according to the supplier’s protocols. The cDNA libraries were then sequenced by Illumina Hiseq 2500 platform. The sequenced raw data were first pre-processed to remove low-quality reads, adaptor sequences and amplification primer. Reads were mapped to Drosophila genome and mapped reads were selected for further analysis. FPKM (Fragments Per Kilobase Of Exon Per Million Fragments Mapped) was used to quantify gene expression.