GSM8012967: Striatal cells, AAV-VEGF-C,; Mus musculus; RNA-Seq
To generate single nuclei transcriptome profiles for mouse brains we used a total of 10 brain samples from adult male mice (8 weeks-old). Five brains from  AAV-mVEGF-C and AAV-CTRL mice were collected,  cryopreserved, pooled, and then processed according to the procedure of nuclei isolation, as previously reported (S. Ma et al. 2022). The brain cell nuclei were isolated according to our previous protocol (10, 13, 41). To avoid anatomical experimental bias for cell nuclei isolation, frozen samples were next finely pulverized to powder in liquid nitrogen with mortar and pestle (Coorstek #60316, #60317). All buffers were ice-cold and all reagents used for consequent nuclear isolation were molecular biology grade unless stated otherwise. 20-30 mg of pulverized tissue was added into 5 ml of ice-cold “lysis buffer”: 320 mM sucrose (Sigma #S0389), 5 mM CaCl2 (Sigma #21115), 3 mM Mg(Ace)2 (Sigma #63052), 10mM Tris-HCl (pH 8; AmericanBio #AB14043), protease inhibitors without EDTA (Roche #11836170001), 0.1 mM EDTA (AmericanBio #AB00502), RNAse inhibitor (80U/ml) (Roche #03335402001), 1mM DTT (Sigma #43186), 0.1% TX-100 (v/v) (Sigma #T8787) nuclease-free water (AmericanBio #AB02123). Reagents: DTT, RNAse Protector, protease inhibitors, TX-100 were added immediately before use. The suspension was transferred to Dounce tissue grinder (15ml volume, Wheaton #357544; autoclaved, RNAse free, ice-cold) and homogenized with loose and tight pestles, 30 cycles each, with constant pressure and without introduction of air. The homogenate was strained through 40-µm tube top cell strainer (Corning #352340) pre-wetted with 1ml “isolation buffer”: 1800 mM sucrose (Sigma #S0389), 3 mM Mg(Ace)2 (Sigma #63052), 10mM Tris-HCl (pH 8) (AmericanBio #AB14043), protease inhibitors without EDTA (Roche #11836170001), RNAse inhibitor (80U/ml) (Roche #03335402001), 1mM DTT (Sigma #43186), nuclease-free water (AmericanBio #AB02123). Additional 9 ml of “isolation buffer” was added to wash the strainer. Final 15 ml of solution was mixed by inverting the tube 10x and carefully pipetted into 2 ultracentrifuge tubes (Beckman Coulter #344059) onto the “isolation buffer” cushion (5 ml) without disrupting the phases. The tubes were centrifuged at 30000 x g, for 60 min at 4 ˚C on the ultracentrifuge (Beckman L7-65) and rotor (Beckman SW41-Ti). Upon end of ultracentrifugation, the supernatant was carefully and completely removed and 100 ul of resuspension buffer (250 mM sucrose (Sigma #S0389), 25 mM KCl (Sigma #60142), 5mM MgCl2 (Sigma #M1028), 20mM Tris-HCl (pH 7.5) (AmericanBio #AB14043; Sigma #T2413), protease inhibitors w/o EDTA (Roche #11836170001), RNAse inhibitor (80U/ml) (Roche #03335402001), 1mM DTT (Sigma #43186), nuclease-free water (AmericanBio #AB02123)) was added dropwise on the pellet in each tube and incubated on ice for 15 minutes. Pellets were gently dissolved by pipetting 30x with 1ml pipette tip, pooled and filtered through 35-µm tube top cell strainer (Corning #352235). Finally, nuclei were counted on hemocytometer and diluted to 1 million/ml with “sample-run buffer”: 0.1% BSA (Gemini BioProducts #700-106P), RNAse inhibitor (80U/ml) (Roche #03335402001), 1mM DTT (Sigma #43186) in DPBS (Gibco #14190). The nuclei samples were placed on ice and taken to Yale Center for Genome Analysis core facility and processed within 15 minutes for snRNA-seq with targeted nuclei recovery of 10,000 nuclei (4 reactions/technical replicates for sum of 40,000 per biological sample), respectively, on microfluidic Chromium System (10x Genomics) by following the manufacturer's protocol (10x Genomics, CG000183_Rev_A), with Chromium Single Cell 3ʹ GEM, Library & Gel Bead Kit v3, (10x Genomics #PN-1000075) and Chromium Single Cell B Chip Kit (10x Genomics #PN1000074), Chromium i7 Multiplex Kit (10x Genomics #PN-120262) on Chromium Controller (10x Genomics). Due to limitations imposed by source RNA quantity, cDNA from nuclei was amplified for 14 cycles Post cDNA amplification cleanup and construction of sample-indexed libraries and their amplification followed manufacturer's directions (10x Genomics, CG000183_Rev_A), with the amplification step directly dependent on the quantity of input cDNA. In order to reach sequencing depth sufficient for characterization of all cells, we targeted 30,000 raw reads per nucleus. Single nucleus libraries were run using paired end sequencing with single indexing on the HiSeq 4000 platform (Illumina) by following manufacturer's instructions (Illumina; 10x Genomics, CG000183_Rev_A). To avoid lane bias, multiple uniquely indexed libraries were multiplexed and distributed over several lanes. The lanes product were then freezed and kept for further analysis in a second run of the same lanes. single nuclei RNA-seq