GSM8503738: APP/PS1 + OSK mPFC, replicate 4, snRNAseq; Mus musculus; RNA-Seq
Mice were sacrificed one day after the last recall session and the mPFC microdissected, flash-frozen in dry ice and finally stored in –80°C until processing. Nuclei were extracted as previously described (Misha). Briefly, tissue of individual animals was kept on ice throughout the protocol and first homogeneized in 600 µl of homogeneization buffer (250 mM sucrose, 25 mM KCl, 5 mM MgCl2, 10 mM Tris⋅HCl, pH 7.4, 1 mM DTT, 1X protease inhibitor, 1 U/µl RiboLock RNAse inhibitor, 0.1% NP-40, in H20), gently filtered through a 40 µm cell strainer and washed with 1500 µl nuclei isolation medium (without NP-40). Samples were centrifuged for 8 min at 1000g and pellet resuspended in 210 µl of isolation medium and then added 290 µl of 50% iodixanol (optiprep, IDM 1X, 1 mM DTT, 1X protease inhibitor 50X, 1 U/µl RiboLock RNAse inhibitor). Nuclei suspension was carefully transferred to a Beckman tube (ref: 344090) and completed with 100 µl of nuclei isolation medium layered on top before ultracentrifugation at 10000g for 22 min using a SW 55 Ti rotor, for debris separation. The nuclei pellet was resuspended in 100 µl of permeabilization buffer (nuclei isolation medium plus 0.01%  Tween, 0.001% Digitonin, 0.01% NP-40, 0.5% BSA), kept on ice for 2 min at 4 °C and finally washed in 500 µl of nuclei isolation buffer plus 0.5% BSA. Samples were centrifuged at 1000g for 10 min at 4°C, pellets resuspended in diluted nuclei buffer (Chromium nuclei isolation kit, 10X Genomics) and nuclei filtered through a 40 µm Flowmi strainer before counting (average yield of 4000 nuclei/µl). Finally, nuclei were processed for Chromium Next GEM Single Cell Multiome ATAC + Gene Expression sequencing 10X Multiome snRNA-seq + snATAC-seq