GSM6216348: 15 days HFHS diet, scRNAseq; Mus musculus; RNA-Seq
ARCs were freshly collected by micropunching (1 mm diameter puncher on 1 mm thick brain section) into cold DMEM/F12 medium (Thermo Fisher Scientific Inc.; #11320033). The samples derived from 6 mice of the same experimental group were pooled together and washed once with cold D-PBS containing 0.05 g/ml of Trehalose (Sigma-Aldrich; #T0167). The buffer was then discarded and the samples incubated for 16 min at 37°C with 200 µl of 4 mg/ml collagenase/dispase solution (Sigma-Aldrich; #10269638001) supplemented with 0.05 g/ml of Trehalose. After the incubation period, the enzymatic solution was carefully removed and the tissue was incubated for 12 min at 37°C with 200 µl of papain/DNAse solution (prepared following steps 2 and 3 from the kit's catalogue; Worthington Biochemical Corporation; #LK003150) supplemented with 0.05 g/ml of Trehalose. The enzymatic solution was then delicately removed and the tissue resuspended in 200 µl of D-PBS supplemented with 0.05 g/ml of Trehalose and RNAseOUT (1/1000; Thermo Fisher Scientific Inc.; #10-777-019), and gently tritured until the solution appeared milky and devoid of visible tissue pieces. The resulting cell suspension was filtered through a 40 µm cell strainer, and cells were pelleted by centrifugation for 8 min at 300 x g and 4°C in 40 ml of D-PBS containing 2% bovine serum albumin (BSA) (Miltenyi BioTec; #130-091-376). Cells were then washed one time by centrifugation (300 x g, 10 min, 4°C) in D-PBS containing 0.04% BSA, and resuspended in 40 µl of the same fresh solution. The final number and viability of the cells was assessed with a hemocytometer after staining with trypan blue. The final volume was adjusted to obtain a concentration of 1000 cells/µl. Chromium Single Cell 3ʹ Reagent Kits v3.1 (10x Genomics, #PN-1000121) was used to prepare libraries, according to the manufacturer's instructions. Libraries were pooled and sequenced according to 10x Genomics' recommendations on an Illumina NovaSeq6000 system with a target read depth of 50000 reads/cell.