GSM8155484: RT00373N snRNA-seq; Homo sapiens; RNA-Seq
To obtain pure and intact nuclear populations, brain tissues were homogenized by a Dounce homogenizer in an ice-cold isolation buffer containing 2M sucrose. All buffers were ice-cold and all reagents used for nuclear isolation were molecular biology grade unless stated otherwise. A total of 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], 10 mM Tris-HCl [pH 8; AmericanBio #AB14043], protease inhibitors without EDTA [Roche #11836170001], 0.1 mM EDTA [AmericanBio #AB00502], RNAse inhibitor [80 U/ml; Roche #03335402001], 1 mM dithiothreitol [DTT; Sigma #43186], and 0.1% TX-100 [v/v; Sigma #T8787]). Reagents DTT, RNAse protector, protease inhibitors, and TX-100 were added immediately before use. The suspension was transferred to a Dounce tissue grinder (15 ml 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 a 40-µm tube top cell strainer (Corning #352340) pre-wetted with 1 ml isolation buffer (1,800 mM sucrose [Sigma #S0389], 3 mM Mg[Ace]2 [Sigma #63052], 10 mM Tris-HCl [pH 8; AmericanBio #AB14043], protease inhibitors without EDTA [Roche #11836170001], RNAse inhibitor [80 U/ml, Roche #03335402001)] and 1 mM DTT [Sigma #43186]. An additional 9 ml of isolation buffer was added to wash the strainer. The final 15 ml of solution was mixed by inverting the tube 10x and carefully pipetted into two ultracentrifuge tubes (Beckman Coulter #344059) onto the isolation buffer cushion (5 ml) without disrupting the phases. The tubes were centrifuged at 30,000 x g for 60 min at 4ºC on an ultracentrifuge (Beckman L7-65) and rotor (Beckman SW41-Ti). Following the ultracentrifugation, the supernatant was carefully and completely removed, and 100 µl of resuspension buffer (250 mM sucrose [Sigma #S0389], 25 mM KCl [Sigma #60142], 5 mM MgCl2 [Sigma #M1028], 20 mM Tris-HCl [pH 7.5; AmericanBio #AB14043; Sigma #T2413], protease inhibitors without EDTA [Roche #11836170001], RNAse inhibitor [80 U/ml; Roche #03335402001], and 1 mM DTT [Sigma #43186]) was added dropwise on the pellet in each tube and incubated on ice for 15 min. Pellets were gently dissolved by pipetting 30x with a 1 ml pipette tip, pooled, and filtered through a 35-µm tube top cell strainer (Corning #352235). Finally, nuclei were counted on a Countess cell counter (ThermoFisher) and diluted to 1 million/ml with sample-run buffer (0.1% bovine serum albumin [Gemini Bio-Products #700-106P], RNAse inhibitor [80 U/ml; Roche #03335402001], and 1 mM DTT [Sigma #43186]) in Dulbecco's phosphate-buffered saline (Gibco #14190). single nuclei multiome (snRNA-seq and snATAC-seq): The nuclei samples were placed on ice and taken to the Yale Center for Genome Analysis core facility, where they were processed with targeted nuclei recovery of 20,000 nuclei per sample on a microfluidic Chromium System (10x Genomics) following the manufacturer's protocol (10x Genomics, Chromium Next GEM Single Cell Multiome ATAC + Gene Expression Reagent Bundle, PN-1000283). Libraries were sequenced with paired-end 150 bp reads on an Illumina NovaSeq 6000 to a target depth of 250 million read pairs per sample.