GSM6632782: SCA1 mouse 2; Mus musculus; RNA-Seq
Frozen or fresh whole cerebellum was placed into 1.5mL tube with 500uL pre-chilled detergent lysis buffer [low sucrose buffer (LSB) (sucrose 0.32M, 10 mM HEPES, 5mM CaCl2, 3mM MgAc, 0.1mM EDTA, 1mM DTT) with 0.1% Triton-X] and the tissue was homogenized using a mechanical homogenizer. A 40um strainer was placed over a pre-chilled 50mL tube and pre-wetted with 1ml LSB. 1mL of LSB was added to the tube containing the crude nuclei in the lysis buffer and mixed gently by pipetting (2-3 times). Crude nuclei prep was then passed over the 40uM strainer into the pre-chilled 50mL tube, washed with 1 ml LSB and centrifuged at 3200g for 10 minutes at 4C.  The pellet was resuspended in 3mL of LSB while gently swirling to remove the pellet from the wall, facilitating resuspension, and left on ice for 2 min. The suspension was transferred to an Oak Ridge tube and nuclei were homogenized in LSB for 15-30s, all while keeping the sample on ice. Using a serological pipette, 12.5mL of density sucrose buffer (sucrose 1M, 10 mM HEPES, 3mM MgAc, 1mM DTT) was layered underneath the LSB homogenate, taking care not to create a bubble which would disrupt the density layer. Samples were then centrifuged at 3200g for 20 minutes at 4C and pelleted nuclei were resuspended in the resuspension solution (PBS, 0.4 mg/ml BSA, 0.2 U/μl RNAse inhibitor). Nuclei were filtered through a 40um pore-size strainer followed by a 30um and 20um pore-size strainers. A small sample of nuclei was pelleted and added to a slide with VectaShield with DAPI to verify single nuclei isolation under fluorescent microscope. Library construction and sequencing were done by University of Minnesota Genomic Center according to the manufacter's instructions (10x Genomics).