GSM8528434: Animal1, SSp, lib1, CYP, ATAC; Mus musculus; ATAC-seq
Nuclear extraction for both mice and primates was performed with density gradient centrifugation according to the protocol described previously with modifications to scale down the reaction volume. Samples were moved from -80 ̊C and thawed on ice for 2 minutes. Tissues were transferred to a dounce homogenizer (Millipore Sigma, D8938-1SET) in homogenization buffer (0.25 M sucrose, 25 mM KCl, 5 mM MgCl2, 10 mM Tris-HCl, pH 8.0, 5 µg/mL actinomycin, 1% BSA, and 0.08 U/µl RNase inhibitor, 0.01% NP40) on ice. Samples were homogenized for 10 strokes with the loose pestle in a total volume of 1 mL, followed by 10 additional strokes with the tight pestle. The tissue homogenate was then passed through a 50 µm filter and diluted 1:1 with working solution (50% iodixanol, 25 mM KCl, 5 mM MgCl2, and 10 mM Tris-HCl, pH 8.0). Nuclei were layered onto an iodixanol gradient after homogenization and ultracentrifuged as described previously. After ultracentrifugation, nuclei were collected between the 30 and 40% iodixanol layers and diluted with resuspension buffer (1xPBS with 1% BSA, and 0.08 U/µl RNase inhibitor). Nuclei are isolated using FACS based on 7-AAD (all nuclei) or 7-AAD and GFP (Projection-TAG+ nuclei). Sorted nuclei are then pooled and diluted to the loading concentration recommneded by the library manuals. Nuclei were processed according to the manufacturer's manual of 10X Genomics Chromium Single Cell Gene Expression 3' V3.1 Assay or 10X Genomics Chromium Single Cell Multiome Assay. snRNA-seq or snMultiome-seq (bombinatorial RNA and ATAC)