title: GSM5604678: Neuron 2-2_KO _scRNA-seq; Homo sapiens; RNA-Seq
library_construction_protocol: Neurons expressing Syn1::GFP were digested using Neurosphere dissociation kit (MACS) according to the manufacturer's instructions and astrocytes expressing GFAP::tdTomato were digested using Papain and DNase I (Worthington) for 30 min at 37℃. Syn1-GFP+ or GFAP-tdTomato+ single cells were sorted using Moflo Astrios (Beckman Coulter) and collected in RNase-free 96-well-plate. An optimized Smart-seq2 protocol was used to construct single-cell RNA-seq libraries while sample pooling and molecular counting were using oligo-d (T) primers comprising 16-bp cell barcode sequence and 9-bp molecular barcode sequence. For compatible with Illumina sequence platform, part of the ISPCR sequence was replaced by read2 sequence in the oligo-d(T) primer. Sorted neuron and astrocytes were denatured in lysis buffer on 72°C for 3min, and put on ice for annealing immediately, then reverse transcribed with template switching oligos and pre-amplified with both ISPCR and read2 primers. cDNA libraries from different cells with different barcodes were pooled together and purified with 0.8x AMPure XP beads. Purified cDNA was fragmented with homemade Tn5 enzyme, and final amplification using Tn5PCR-7 primers and Tn5PCR-5 primers (Nextera). Amplificated cDNA libraries were purified twice with AMPure XP beads.