GSM8385020: Dopaminergic neuron nuclei, Lrrk2 G2019S mutants, sample 1; Mus musculus; RNA-Seq
A 2mm thick block of ventral midbrain tissue was dissected out from each mouse and collected for isolation. Tissue was dounce homogenized in a nuclear extraction buffer (10mM Tris, 146mM NaCl, 1mM CaCl2, 21mM MgCl2, 0.1% NP-40, 40u/mL Protector RNAse inhibitor (Roche 3335399001). Dounce homogenizer was washed with 4mL of a washing buffer (10mM Tris, 146mM NaCl, 1mM CaCl2, 21mM MgCl2, 0.01% BSA, 40U/mL Protector RNAse inhibitor) and filtered through a 30uM cell strainer. After three rounds of washing by centrifugation at 500g for 5 minutes, nuclei pellets were resuspended resuspension buffer (10mM Tris, 146mM NaCl, 1mM CaCl2, 21mM MgCl2, 2% BSA, 0.02% Tween-20) and filtered through a 20uM strainer. This nuclei suspension was loaded onto a MACSQuant Tyto HS chip and diluted with 1x PBS. Nuclei were sorted using gates set for isolation of GFP+ singlet nuclei. The cDNA and library were generated using the Chromium Next GEM Single Cell 3' Reagent Kits v3.1 (10X Genomics, PN-1000286) and Dual Index Kit TT Set A (10X Genomics, PN-1000215) according to the manufacturer's manual with following modification: PCR cycle used for cDNA generation was 16 and the resulting PCR products was size-selected using 0.8X SPRI beads instead of 0.6X SPRI beads as stated in protocol.