GSM7029484: M1 nuclei, control, mouse1, deep snRNAseq; Mus musculus; RNA-Seq
All brains were serially sectioned in ice-cold, carbogen-bubbled ACSF on a VT1000S vibratome (Leica) with polytetrafluoroethane-coated razor blades (Ted Pella) at 0.15 mm/sec and 100 Hz, dividing the whole cerebrum into 400 µm coronal slices. Each slice between 0 and 1.2 mm anterior to bregma (2-4 slices per animal) was dissected under a stereomicroscope using a microsurgical stab knife to cut out the caudal forelimb area (CFA) in the motor cortex of the right hemisphere (~1 to 2 mm from the midline). All extracted tissue was recovered in ice-cold, nuclease-free, 0.22 µm sterile-filtered cryoprotective nuclear storage buffer, composed of 0.32 M sucrose, 5 mM CaCl2, 3 mM magnesium acetate, 10 mM Trizma hydrochloride buffer (pH 8.0), 1 mM dithiothreitol, 0.02 U/µl SUPERase•In RNAse Inhibitor (Invitrogen), and 1X cOmplete Protease Inhibitor Cocktail with EDTA (Roche). Tissue was then flash frozen using a metal CoolRack M90 (Biocision) pre-chilled to -80˚C and stored at -80˚C until nuclear isolation. Single nucleus RNA-seq was provided by the Center for Epigenomics, UC San Diego using the Droplet-based Chromium Single-Cell 3' solution (10x Genomics, v3.1 chemistry) [5]. Nuclei preparation was adapted from Lacar et al. 2016 (PMID: 27090946). The flash frozen mouse motor cortex tissues were thawed on ice for ~30-50 minutes before centrifugation for 3 minutes at 100 rcf in a 4 °C, swinging bucket centrifuge with setting run 3/3 (5920, Eppendorf). The supernatant was removed and the tissue was resuspended in 1 ml of  douncing buffer consisting of 0.25M sucrose (S1888, Sigma), 25mM KCl (AM9610G, Invitrogen), 5mM MgCl2 (194698, Mp Biomedicals Inc.), 10 mM Tris-HCl pH 7.5 (15567027, Thermo Fischer Scientific), 1mM DTT (D9779, Sigma), 1X protease inhibitor (05056489001, Roche), 0.1% Triton X-100 (T8787-100ML, Sigma), and 0.2 U/ul RNasin RNase inhibitor (PAN21110, Promega) in molecular biology grade water (46000-CM, Corning). The tissue was then transferred to a dounce homogenizer on ice and dounced 25 times with a loose plunger and 25 times with a tight plunger. Suspension was then passed through a Celltrix 30 μM filter (04-004-2326, Sysmex) and washed with 300 ul of douncing buffer before being centrifuged for 10 minutes at 1000 rcf. Pellet was then washed with an additional 1 ml of douncing buffer without Triton X-100 and centrifuged again for 10 minutes at 1000 rcf. Supernatant was then discarded, and the pellet was resuspended in 500 ul of sort buffer consisting of 1 mM EDTA (15575020, Invitrogen), 0.2 U/ul RNasin, and 1% BSA in PBS before staining with 3 μM DRAQ7 (#7406S, Cell Signaling Technology). Nuclei were then incubated on ice for 10 minutes and ~20-30,000 nuclei were sorted using a 100 μm chip in an SH800 sorter (Sony) into 50 ul of collection buffer consisting of 1 U/ul RNasin and 5% BSA in PBS. Samples were then centrifuged for 15 minutes at 1000 rcf; the supernatant was removed, leaving behind ~20 ul, and an additional 25 ul of reaction buffer consisting of 0.2 U/ul RNasin and 1% BSA in PBS was added for a total volume of ~45-50 ul. The prepared nuclei were visually inspected and manually counted using a hemocytometer before loading 15,000 onto a Chromium Controller for 10x GEM generation in the Single Cell 3' v3.1 kit (1000268, 10x Genomics).  Libraries were generated using the Chromium Single Cell 3′ Library Construction Kit v3.1 (1000190, 10x Genomics) according to manufacturer specifications. CDNA was amplified for 12 PCR cycles. SPRISelect reagent (Beckman Coulter) was used for size selection and clean-up steps. Final library concentration was assessed by Qubit dsDNA HS Assay Kit (Thermo-Fischer Scientific) and fragment size was checked using Tapestation High Sensitivity D1000 (Agilent) to ensure that fragment sizes were distributed normally about 500 bp.