GSM5060358: Learned_Motor Learning Paradigm Rep2; Mus musculus; RNA-Seq
Whole cerebellum or anterior lobules (anterior dorsal cerebellar vermis, ADCV) microdissected from mice upon different training conditions (Activation rotarod (baseline, activation), accelerating motor learning (Baseline, learning, learned), and associated motor learning (Baseline, learning and learned) were rapidly transferred to a chilled dounce homogenizer and dounced in 1.5 ml homogenization buffer containing 0.25 M sucrose, 150 mM KCl, 5 mM MgCl2, 20 mM Tricine-KOH pH 7.8, 1 mM DTT, 0.15 mM spermine, 0.5 mM spermidine, protease inhibitor to release nuclei. Each sample were mixed with 1.5 ml 50% iodixanol (Sigma) and layered onto 29% iodixanol. The gradient was centrifuged at 10,000 g for 30 mins at 4°C. Nuclei were diluted in 10% BSA with Dyecycle Ruby for FACS. Dyecycle Ruby was used for singlet and GFP for positive was sorted for Purkinje nuclei. After counting and analyzing nuclei integrity, all samples were subjected to library construction by using Chromium Single Cell 3' Reagent Kit V3 (10X Genomics). Single nuclei libraries were prepared for sequencing using standard protocols