GSM8144099: STSC3; Mus musculus; RNA-Seq
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 FACS Aria Fusion sorter with a 70 μM nozzle at 22.5 PSI sheath pressure. DAPI+/mCherry+ rabies-infected nuclei were sorted into 0.2ml Eppendorf PCR tubes and immediately loaded onto the 10X Genomics Chromium Controller. Biological replicates were processed on different days. For 10x processing, we used Chromium Next GEM single-cell 3' Kit (v3.1 Dual Index) and Chromium Next GEM single-cell 3' LT Kit (v3.1 Dual Index) (10x Genomics, PN-1000127 and PN-1000325). 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. Pooled libraries were sequenced on Illumina NovaSeqTM 6000 Sequencing System (S4)