GSM6190912
Single nuclei preparations were performed from V1 dissected regions and nuclei sorted for NeuN-positive nuclei. Single nuclei preparations were performed as follows: the frozen brain tissues were transferred to pre-chilled dounce homogenizers with 1ml NIM buffer (0.25M sucrose, 25mM KCl, 5mM MgCl2, 10mM Tris-HCl (pH7.4), 1mM DTT (Sigma 646563), 10ul of protease inhibitor (Sigma P8340), 1.5ul of RNasin Plus RNase inhibitor (Promega, N2611)), 0.1% Triton X-100, and 10 uM DAPI, and gently homogenized on ice with ice-cold pestles 10 - 15 times. The homogenate was transferred to pre-chilled microcentrifuge tubes and centrifuged at 3000 rpm for 8 min at 4°C to pellet the nuclei. The supernatant was aspirated, the pellet gently resuspended in ice-cold 1ml NIM buffer, and again centrifuged at 3000 rpm for 8 min at 4°C. The pellet was then resuspended in 450ul of nuclei storage buffer (0.25M sucrose, 5mM MgCl2, 10mM Tris-HCl (pH7.4), 1mM DTT, 9ul of Protease inhibitor), and filtered through a 40μM cell strainer. The sample was incubated with 50ul of nuclease-free BSA to prevent nuclei clumping. Fluorescence-activated nuclei sorting of single nuclei was performed using a BD Influx sorter with a 70 μM nozzle at 22.5 PSI sheath pressure. DAPI+/mCherry+ rabies-infected nuclei were sorted into 1.5ml Eppendorf tubes and immediately loaded onto the 10X Genomics Chromium Controller. For 10x processing, we used Chromium Next GEM single-cell 3' Kit v3.1 (10x Genomics, PN-1000128). We followed the manufacturer's instructions for single-cell capture, barcoding, reverse transcription, cDNA amplification, and library construction. We targeted sequencing depth of 100,000 reads per cell. Libraries were sequenced on both Illumina NextSeq550 and NovaSeq6000 and raw read (fastq) files from both sequencing platforms were combined and aligned to the mouse pre-mRNA reference transcriptome (mm10) using the 10x Genomics CellRanger pipeline (version 5.0.). Intronic reads were included in expression quantification using the include-introns parameter snRNA-Seq