title: GSM8527127: SNr; Mus musculus; RNA-Seq
library_construction_protocol: 9 12-week old C57BL/6JN mice were used. Animals were euthanized in a CO2 chamber and decapitated, after which the brains were dissected out from the skull and submerged in ice-cold aCSF (92 mM NMDG, 2.5 mM KCl, 1.25 mM NaH2PO4, 30 mM NaHCO3, 20 mM HEPES, 25 mM glucose, 5 Na-ascorbate, 3 mM Na-pyruvate, 0.5 mM CaCl2·2H2O, and 10 mM MgSO4·7H2O). Coronal brain slices (400 µm thick) were cut using a vibrating-blade microtome (V1200S, Leica, USA) at 4°C. The SNr subregion was microdissected out in aCSF under a fluorescent dissection microscope (Leica M165FC) and transferred to microcentrifuge tubes. Excess aCSF was aspirated before flash-freezing the tubes on dry ice. Single nuclei preparations were performed pooling tissue from 3-4 mice, following the Allen Institute's published V1 protocol. Briefly, each pooled sample was homogenized in a chilled 2 mL dounce (Sigma D8938) using 3.5 ml of freshly prepared homogenization buffer containing 3.375 ml of NIM (10 mM Tris-HCl, 250 mM sucrose, 25 mM KCl, 5mM MgCl2, 10mM Tris-HCl ), 3.5 µl of 100 mM DTT stock (Promega P1171), 70 µl of 50X Protease inhibitor cocktail (Promega G6521), 17.5 µl of 40 U/µl RNAsin Plus (Promega N2615), and 35 µl of 10% Triton X-100 (Sigma T8787). The homogenate was strained through a 30 µm cell strainer (Miltenyi Biotec 130-098-458) and centrifuged at 900 x g for 10 minutes at 4°C. After centrifugation, the pellet was resuspended in 500 µl of freshly prepared blocking buffer containing 410 µl nuclease-free water, 50 µl 10X PBS, 40 µl of 10% OmniPur BSA (Sigma 2905), and 2.5 µl of 40 U/µl RNAsin Plus. The nuclear resuspension as subsequently incubated on ice for 15 minutes. The suspension was labeled with Mouse anti-NeuN-488 to enrich for neuronal cells (1:500, Millipore Sigma), incubated on ice for 30 minutes, then centrifuged at 400 x g for 5 minutes. The supernatant was resuspended in 500 µl of blocking buffer, then labeled with 0.5 µl of DAPI. Prior to DAPI labeling, 5 µl of nuclear suspension was removed and labeled with 5 µl of Trypan Blue and loaded into a disposable hemocytometer slide (Invitrogen C10228) for quantification of yield and visual inspection of nuclear quality. Fluorescence-activated nuclei sorting (FANS) of single nuclei was performed using the Beckman Coulter MoFlo Astrios EQ. Cell analysis and sorting were performed on a Beckman Coulter MoFlo Astrios EQ.  These experiments required the use of the 405 nm (80 mW) laser for fluorescent excitation of the DAPI labeled nuclei and the 488 nm (200 mW) laser for forward, side scatter detection and GFP excitation. The sample and collection area were cooled to ~ 7° C. DAPI was detected with a 448 /59 band-pass filter. The PMT was set at 341V with an amplifier gain of 3. GFP was detected with a 513 /26 band-pass filter. The PMT was set at 360V with an amplifier gain of 3. The PMT was set at 310V with an amplifier gain of 4. DAPI+/GFP+ nuclei were collected into an Eppendorf tube containing blocking buffer. Single-nucleus cDNA libraries were constructed according to the user guide of Chromium Single Cell 3' Reagents Kit v3 (10× Genomics, Pleasanton, CA).