GSM7429766: CTX_adult_ast_4; Mus musculus; RNA-Seq
Adult (4mo) and aged (2yo) male C57BL/6 mice (3 mice/group) were obtained from Jackson laboratories (adult) or the NIA (aged) and housed for 3 weeks. Mice were anesthetized with ketamine (10mg/ml) and xylazine (2mg/ml) solution delivered intraperitoneally, decapitated and cortex and cerebella were dissected. Dissected tissues were placed in 4 mL of 3% glyoxal solution for 15 min on ice. After that, samples were washed 3 times in PBS1X and nuclei were obtained after tissue homogenization with a dounce homogenizer followed by iodixanol gradient. Nuclei suspensions were stained with Hoetsch, anti-Sox9 and anti-NeuN antibodies, and with fluorescently-conjugated secondary antibodies. Sox9+NeuN- astrocyte and NeuN+ neuron nuclei were FACS-purified with an Aria II Fusion (BD). RNA was extracted with Rnaeasy Micro Kit (Qiagen) following manufacturer's instructions. RNA quality and quantity were determined with TapeStation (High sensitivity RNA tape) following manufacturer's instructions. 2 ng per sample were used to construct libraries with the Illumina Stranded Total RNA prep (with Ribo-zero plus) sequencing kit, following manufactuter's instructions.