Single-cell RNA-seq of Mouse Neurons

Total RNA was isolated from flow-sorted neurons using Trizol LS (Invitrogen) and the RNA Clean and Concentrator-5 kit (Zymo Research). RNA concentration and quality was determined using Quantifluor RNA system (Promega) and Fragment analyzer (Advanced Analytics). Samples were converted to cDNA, depleted of rRNA transcripts, and amplified using the TRIO RNA-seq kit for mouse (Nugen Inc). Single-end sequencing was performed on a Nextseq 550 (Illumina) for 76 cycles. Read quality was assessed using FASTQC (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/) and adapters were trimmed using Trimmomatic (Bolger et al., 2014 (link)). Filtered and trimmed reads were aligned to the mouse genome (mm10, UCSC) using Tophat2 (v2.1.1) (Kim et al., 2013 (link)).