title: GSM8875867: Human, Caudate Nucleus, rep 4; Homo sapiens; RNA-Seq
library_construction_protocol: Nuclei for snRNA-seq were isolated from bat putamen and caudate brain tissue. Briefly, the tissue was homogenized in 500 µL of ice-cold Nuclei EZ lysis buffer (EZ PREP NUC-101, Sigma) using a pre-chilled glass Dounce homogenizer. Debris was removed via density gradient centrifugation using Nuclei PURE 2 M sucrose cushion solution and Nuclei PURE sucrose cushion buffer (Nuclei PURE prep isolation kit, no. NUC201-1KT, Sigma Aldrich). These two solutions were mixed in a 9:1 ratio, and 500 µL of the sucrose solution was added to a 2-ml Eppendorf tube. The homogenized nuclei suspension was mixed with 900 µL of sucrose cushion solution by pipetting 10 times, resulting in a 1,400 µL volume, which was carefully layered over the sucrose buffer without mixing. The samples were centrifuged at 13,000 x g for 45 min at 4 °C, and all but 100 µL of supernatant was discarded. Nuclei were resuspended in 300 µL of nucleus suspension buffer (NSB), consisting of 1× PBS, 2% BSA (no. AM2618, Thermo Fisher Scientific), and 0.4 U µL −1 RNAse inhibitor (no. AM2694, Thermo Fisher Scientific). Samples were then centrifuged at 550 x g for 5 min at 4 °C, and all but 50 µL of supernatant was discarded. The nuclear pellet was resuspended in NSB and filtered through a 40-μm Flowmi cell strainer (Bel-Art Products, #H13680-0040) into a new tube. The nuclei concentration was measured using Hoechst and decreased to 1,000 nuclei per µL with NSB if necessary. Cellular nuclei for snRNA-seq were isolated from the putamen and caudate nuclei of human, chimpanzee, and rhesus macaque tissues. Briefly, the tissue was homogenized in 750 µL of ice-cold NP40 lysis buffer (10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl₂, 0.1% Nonidet P40 Substitute, 1 mM DTT, 0.2 U/µL RNAse inhibitor) using a pre-chilled glass Dounce homogenizer. An additional 750 µL of ice-cold NP40 lysis buffer was added, and the mixture was pipette-mixed during a 5-minute incubation on ice. Nuclei were centrifuged at 500 x g for 5 minutes at 4°C, resuspended in 1 mL of ice-cold lysis buffer, and incubated on ice for 5 minutes with gentle pipette mixing throughout. The nuclei were then centrifuged again at 500 x g for 5 minutes at 4°C, and all but 50 µL of supernatant was discarded. Next, 500 µL of nucleus suspension buffer (NSB) containing 1× PBS, 1% Ultrapure BSA (no. AM2618, Thermo Fisher Scientific), and 0.2 U/µL RNAse inhibitor (no. AM2694, Thermo Fisher Scientific) was added, and the sample was incubated for 5 minutes on ice without mixing. The pellet was resuspended, centrifuged again at 500 x g for 5 minutes at 4°C, and all but 50 µL of supernatant was discarded. The nuclei were then resuspended in 500 µL of NSB and filtered through a 70-μm Flowmi cell strainer (no. H13680-0070, Bel-Art) into a 1.5 mL tube. Droplet-based snRNA-seq libraries were prepared using the Chromium Single Cell 3′ v3.1 (10x Genomics) kit, following the manufacturer's protocol. Libraries were sequenced on an Illumina NovaSeq 6000.
identifiers.namespace: GEO
identifiers.id: GSM8875867_r1