GSM9066377: A2022 5XFAD IDOL WT; Mus musculus; RNA-Seq
Nuclei from frozen posterior cortical tissue from female WT and Neu cKO (N=2/genotype) were isolated using the Singulator 100 (S2 Genomics)20. Briefly, frozen brain samples were added to precooled Nuclei Isolation Cartridges (S2 Genomics, 100-063-623) along with RNAse inhibitor (Roche, 03335402001) and loaded into the Singulator 100 to run on Standard Nuclei Isolation Protocol v2. The nuclei suspensions were transferred to conical tubes and pelleted at 500 g for 5 minutes at 4 °C. Once pelleted, the supernatant was aspirated, and the pellets were resuspended in 20% Percoll (100-253-628; S2 Genomics) diluted in Nuclear Storage Reagent (NSR; 100-063-623; S2 Genomics). The samples were centrifuged at 700 g for 8 minutes at 4 °C with the brake off. The nuclei were washed with the Resuspension Buffer (2% BSA in NSR), spun down at 500 g for 5 min at 4 °C, and resuspended in the Resuspension Buffer. The number and viability of the cells were measured using trypan blue staining and observed with an EVOS XL Core microscope. Nuclei suspensions were processed in a single batch by the 10X Chromium. Each single cell mix was loaded into a Chip G and run on the Chromium Controller for GEM generation and barcoding. Sample processing and library preparation were performed according to the manufacturer's instructions using the Chromium Next GEM Single Cell 3' v3.1 dual index kit (10X Genomics) and SPRIselect paramagnetic bead-based chemistry (Beckman Coulter Life Sciences). The cDNA and library quality were assessed using the 2100 Bioanalyzer and a High Sensitivity DNA kit (Agilent Technologies). The final library concentration was determined using a QuBit Fluorometer and the dsDNA HS assay kit (Thermo Fisher Scientific). Sequencing was carried out on a NovaSeq 6000 (v1.5 S2; Illumina) with 28-10-10-91 read setup.
GEO
GSM9066377_r1