GSM6506795: R62+hGPC 1; Mus musculus; RNA-Seq
To prepare samples for single-nucleus RNA sequencing, dissected striata were pooled and placed in 1 mL of ice-cold Nuclei Isolation Buffer (NIB), consisting of 10 mM HEPES pH 7.9, 10 mM KCl, 0.5 mM Spermidine, 0.10% IGEPAL, 1x cOmplete EDTA-free protease inhibitor cocktail (Roche), and 0.2 U/uL Protector RNase Inhibitor (Roche). Tissue was manually triturated with a P1000 pipette, then incubated for 10 minutes on ice. Samples were then centrifuged for 3 minutes at 600g/rcf. The nuclei pellets were resuspended in 500 uL NIB, then incubated for 3 more minutes on ice. Nuclei were centrifuged for 3 minutes at 600g/rcf, then resuspended in DMEM/F-12. Nuclei were then incubated with 7-Aminoactinomycin D (7-AAD) (Invitrogen) added 1:1000, then sorted on the BD FACSAria Fusion (BD Biosciences). Sorted nuclei were counted and loaded onto a 10X Genomics Chromium controller to generate single-nucleus gel beads in emulsion (10X Genomics). Libraries were prepared using 10X Genomics' Chromium Single Cell 3' Reagent Kit v3.1.