title: GSM8824189: S2 Genomics_Singulator100_Sample_1; Mus musculus; RNA-Seq
library_construction_protocol: Centrifugation-based nuclei isolation Frozen anterior cortex tissue was added to a 2 mL tube with 1 mL of ice-cold EZ nuclei suspension buffer (Sigma, EZ PREP NUC-101). The tissue was dissociated with gentle, manual pipetting. The dissociated tissue was transferred into a 15 mL conical tube and an additional 6 mL of ice-cold EZ nuclei suspension buffer was added. The solution was incubated at 4oC for 10 minutes while rotating. After rotating, the sample was centrifuged at 500×g for 5 minutes at 4oC. The supernatant was removed, and the pellet was resuspended in 1 mL of ice-cold EZ nuclei suspension buffer with gentle pipetting. An additional 6 mL of EZ nuclei resuspension buffer was added. The solution was incubated at 4oC for 10 minutes while rotating. After rotating, the sample was centrifuged at 500×g for 5 minutes at 4oC. The supernatant was removed. 1 mL of Nuclei Wash and Resuspension buffer (NWR Buffer) (1x PBS, 2% Bovine Serum Albumin (BSA), and 2U/μl RNAse inhibitor (Millipore Sigma, 3335399001)) was added and mixed with gentle pipetting. An additional 6 mL of NWR Buffer was added. The sample was centrifuged at 500×g for 5 minutes at 4oC. This wash step with NWR Buffer was repeated four times. After the last wash, the nuclei were resuspended in 1mL NWR buffer then another 6 mL was added. A 70-μm and 30-μm cell strainer were pre-wet with LS Column Calibration Buffer (1x PBS, 0.5% BSA). The nuclei were filtered through the 70 μm strainer and then the 30 μm strainer to remove clumps. The remaining solution was centrifuged at 500×g for 5 minutes at 4oC. The supernatant was removed, and the pellet was resuspended in 1 mL of NWR Buffer. 500 μL of the resuspended nuclei were added to an Eppendorf tube containing 900 μL of Sucrose Cushion Buffer (Nuclei PURE 2M Sucrose Cushion Solution (Sigma, S9308), 10% Nuclei PURE Sucrose Cushion Buffer (Sigma, S9058)). The mixture was carefully pipetted together. In a separate Eppendorf Tube, 500 μL of Sucrose Cushion Buffer was added. To create the sucrose gradient, the 1400 μL of nuclei suspension in Sucrose Cushion Buffer was added carefully to the top of the Eppendorf tube containing 500 μL of Sucrose Cushion Buffer with no mixing. The sucrose gradient was centrifuged at 13,000×g for 45 minutes at 4oC. After centrifugation, the supernatant was almost completely removed, leaving approximately 100 μL in the tube. The pellet was resuspended in 1 mL of NWR Buffer with gently pipette mixing. The solution was filtered through a 40 μm cell strainer to remove any leftover debris. Column-based isolation Nuclei suspensions were prepared according to 10x Chromium Nuclei Isolation Kit instructions, with all steps performed on ice[42]. Frozen anterior cortex tissue was added to a pre-chilled Sample Dissociation Tube (10x Genomics, 2000564). 200 μL of Lysis Buffer (10x Genomics, 2000558) was added to the tube and the tissue was dissociated with a pestle. 300 μL of additional Lysis Buffer was added and the sample was incubated on ice for 10 minutes. The solution was then added to a Nuclei Isolation Column (10x Genomics, 2000562) in a Collection Tube (10x Genomics, 2000563). The column was centrifuged at 13,000×g for 20 seconds at 4oC. The flowthrough was quickly vortexed. The solution was then centrifuged at 500×g for 3 minutes at 4oC. The supernatant was removed, and the pellet was resuspended in 500 μL of Debris Removal Buffer (10x Genomics, 2000560). The suspension was centrifuged at 700×g for 10 minutes at 4oC. The supernatant was removed and resuspended in 1 mL of Wash and Resuspension Buffer (1x PBS, 10% BSA, 2.5% RNAse Inhibitor (10x Genomics, 2000565)), then centrifuged at 500×g for 5 minutes at 4oC. This wash step was repeated once more. The supernatant was removed, and the nuclei suspension was resuspended in 50 μL of Wash and Resuspension Buffer by gentle pipette mixing. Machine-assisted nuclei isolation Nuclei were isolated using the Singulator 100 System, with all steps performed on ice[43]. Before isolation, nuclei isolation cartridges (S2 Genomics, #100-063-287) were pre-cooled to -25°C, and the Singulator was pre-chilled to ensure optimal processing conditions. For each sample, the frozen anterior cortex was placed into a pre-cooled nuclei isolation cartridge along with 75 μL of RNAse inhibitor (Millipore Sigma, 3335399001). The sample was processed using the Standard Nuclei Isolation Protocol. The resulting 3 mL nuclei suspension was transferred to a 15 mL conical tube and centrifuged at 500×g for 5 minutes at 4°C to pellet the nuclei. The supernatant was aspirated, and the pellet was resuspended in 3 mL of 20% Nuclei Debris Removal Stock Solution (S2 Genomics, #100-253-628) in Nuclear Storage Reagent (S2 Genomics, #100-063-623). The suspension was centrifuged for 8 minutes at 700×g and 4°C with a decreased brake setting (3 out of 10, Sorvall Legend X1R Centrifuge (ThermoFisher)). The tubes were carefully removed without disturbing the gradient, and the gradient was aspirated from just below the meniscus using a P1000 pipette to remove myelin debris, which was visible as a haze or flake at the top. Finally, the nuclei were resuspended in 3 mL of resuspension buffer (2% BSA in Nuclear Storage Reagent). Nuclei concentration and viability were quantified with trypan blue staining and observed with an EVOS XL Core microscope. All nuclei suspensions were processed by the 10x Chromium. Each nuclei suspension was counted and diluted to 1,000 nuclei per uL then loaded into a Single Cell Chip G and run on the Chromium Controller for GEM generation and barcoding. Sample processing and library preparation were performed according to the manufacturer's instructions using the Chromium Next GEM Single Cell 3′ v3.1 dual index kit (10x Genomics) and SPRIselect paramagnetic bead-based chemistry (Beckman Coulter Life Sciences).  The cDNA and library quality were assessed using the 2100 Bioanalyzer and a High Sensitivity DNA kit (Agilent Technologies). The final library concentration was determined using a QuBit Fluorometer and the dsDNA HS assay kit (Thermo Fisher Scientific).