title: GSM8703604: RA019, Ventral midbrain nuclei, MitoPark 24wks; Mus musculus; RNA-Seq
library_construction_protocol: For nuclei isolation, frozen tissues were placed into a homogenization buffer that consisted of 10mM Tris pH 8.0, 250mM Sucrose, 25mM KCl, 5mM MgCl2, 0.1% Triton-X 100, 1X Protease inhibitor, 0.5% RNasin Plus RNase inhibitor and 0.1mM DTT. Tissues were placed into a 2ml dounce homogenizer and homogenized using 10 strokes of the loose dounce pestle followed by 10 strokes of the tight pestle to liberate nuclei. Homogenate was strained through a 30μm cell strainer and centrifuged at 900xg for 10 minutes to pellet nuclei. Nuclei were then resuspended in blocking buffer containing 1X PBS supplemented with 0.8% nuclease-free BSA and 0.5% RNasin Plus RNase inhibitor. Prior to fluorescence-activated nuclei sorting (FACS), DAPI was applied to nuclei suspensions at a final concentration of 0.1μg/ml and nuclei suspensions were filtered through a 30μm nylon mesh to remove aggregates. Libraries were prepared according to the manufacturer's protocol (10x Genomics Chromium Single Cell 3' Reagent Kit v3).