GSM9305634: young_male_hypothalamus; Mus musculus; RNA-Seq
The anterior hypothalamus region containing the POA (Bregma A/P +0.5 to -0.6 mm) were dissected together from 5 young wild-type male mice (Age 3m) and 5 aged mice (Age 22m). Dissected tissues from 5 mice were pooled for each sample, dounce homogenized on ice and centrifuged at 500g for 10 min at 4 °C. Pellets were resuspended and spun in an iodixanol gradient at 3000g for 1hr, 4 °C.  Myelin was removed using Myelin Removal Beads II (Miltenyi Biotec, Cat#130-096-733) using LS columns (Miltenyi Biotec, Cat#130-042-401) according to the manufacturer's instructions and the extracted cell layer was resuspended in 1% BSA solution with RNAse inhibitor. Nuclei were pelleted at 500g for 10 minutes at 4 °C before final resuspension. The concentration of nuclei was adjusted as per 10x chromium chip requirements.  Supernatants were removed and passed through a 70-μm filter then through a 20-μm filter (MACS Smartstrainer) to remove large debris. DAPI was added to the suspensions and nuclei were separated by FACS. The resulting suspensions were loaded into a 10x Genomics Chromium single-cell chip at a concentration of about 700-1200 nuclei per μl with the aim of sequencing 10000 nuclei per sample. The libraries were sequenced on an Illumina NextSeq500 instrument using instructions provided by 10x Genomics. Paired-end sequencing with read lengths of 150 nt was performed for all samples.