GSM7156229
[nuclei preparation protocol]  Brain tissue was pulverized using a mortar and pestle on dry ice and pre-chilled with liquid nitrogen. Pulverized brain tissue was resuspended in 1mL of chilled NIM-DP-L buffer (0.25M sucrose, 25mM KCl, 5mM MgCl2, 10mM Tris-HCl pH 7.5, 1mM DTT, 1X Protease Inhibitor (Pierce), 1U/μL Recombinant RNase inhibitor (Promega, PAN2515), and 0.1% Triton X-100). Tissue was Dounce homogenized with a loose pestle (5-10 strokes) followed by a tight pestle (15-25 strokes) or until solution was uniform. Nuclei were filtered using a 30μm CellTrics filter (Sysmex, 04-0042-2316) into a LoBind tube (Eppendorf, 22431021) and pelleted (1000 rcf, 10 min at 4˚C) (Eppendorf, 5920 R). Pellet was resuspended in 1 mL NIM-DP buffer (0.25M sucrose, 25mM KCl, 5mM MgCl2, 10mM Tris-HCl pH 7.5, 1mM DTT, 1x Protease Inhibitor, 1U/μL Recombinant RNase inhibitor) and pelleted (1000 rcf, 10 min at 4˚C). Pelleted nuclei were resuspended in 400uL 2μM 7-AAD (Invitrogen, A1310) in Sort Buffer (1mM EDTA, 1U/μL Recombinant RNase inhibitor, 1X Protease Inhibitor, 1% fatty acid-free BSA in PBS). 120,000 nuclei were sorted (Sony, SH800S) into a LoBind tube containing Collection Buffer (5U/uL Recombinant RNase inhibitor, 1X Protease Inhibitor, 5% fatty acid-free BSA in PBS). 5X Permeabilization Buffer (50mM Tris-HCl pH 7.4, 50mM NaCl, 15mM MgCl2, 0.05% Tween-20, 0.05% IGEPAL, 0.005% Digitonin, 5% fatty acid-free BSA in PBS, 5mM DTT, 1U/μL Recombinant RNase inhibitor, 5X Protease Inhibitor) was added for a final concentration of 1X. Nuclei were incubated on ice for 1 minute, then centrifuged (500 rcf, 5min at 4C). Supernatant was discarded and 650uL of Wash Buffer (10mM Tris-HCl pH 7.4, 10mM NaCl, 3mM MgCl2, 0.1%.Tween-20, 1% fatty acid-free BSA in PBS, 1mM DTT, 1U/μL Recombinant RNase inhibitor, 1X Protease Inhibitor) was added without disturbing the pellet followed by centrifuging (500 rcf, 5 min at 4˚C). Supernatant was removed, and the pellet was resuspended in 7uL of 1X Nuclei Buffer (Nuclei Buffer (10x Genomics), 1mM DTT, 1 U/μL Recombinant RNase inhibitor). 1 μL of nuclei was diluted in 1X Nuclei Buffer, stained with Trypan Blue (Invitrogen, T10282) and counted. 16-20k nuclei were used for tagmentation reaction and controller loading and libraries were generated following manufacturer's recommended protocol (https://www.10xgenomics.com/support/single-cell-multiome-atac-plus-gene-expression). 10x multiome ATAC-seq and RNA-seq libraries were paired-end sequenced on NextSeq 500 and NovaSeq 6000 to a depth of ~50,000 reads per cell for each modality.