GSM6246115: AD_SPM_1; Mus musculus; RNA-Seq
Mouse hemispheres were harvested from male mice at the age of 180 days and immediately snap frozen in liquid nitrogen and stored at -80°C until further processing for nuclei isolation. Nuclei were isolated from a single mouse hemisphere in 2 ml of pre-chilled EZ PREP lysis buffer (NUC-101, Sigma) using a glass Dounce tissue grinder (D8938, Sigma) (25 strokes with pastel A and 25 strokes with pastel B) followed by incubation for 5 minutes on ice with additional 6 ml of EZ PREP buffer. During incubation, 1 µM DAPI was added to the homogenate and subsequently filtered through a 35 µm strainer. Intact nuclei were sorted with a BD FACSAriaIII with a 70 µm configuration into 1.5 ml-Eppendorf tubes with 40 µl of 4 % BSA in PBS and RiboLock RNase Inhibitor (25 U/µl, EO0381, ThermoFisher). A FSC/SSC based gate was used to exclude debris followed by exclusion of damaged nuclei in a DAPI-A/DAPI-H (see Supplementary Fig. 5a). 150,000 events were sorted for each sample. The concentration of sorted nuclei was determined based on brightfield images and DAPI fluorescence using a Neubauer counting chamber and a Leica DMi8 microscope. Single nuclei libraries were generated according to the Chromium Next GEM Single Cell 3ʹ Reagent Kits v3.1 User Guide (CG000204) by 10x Genomics. Briefly, a droplet emulsion was generated in a microfluidic chip followed by barcoded cDNA generation inside these droplets. Purified and amplified cDNA was then subjected to library preparation and sequenced on a NovaSeq 6000 instrument (Illumina) to a median depth of 45-90k reads per cell.