title: Single nucleus multiome assay (RNA+ATAC-seq) of Homo sapiens: postmortem brain (caudate nucleus)
design: Nuclei Isolation for Single-cell Multiome  200 fresh-frozen post-mortem caudate brain samples were utilized in the assay. The 200 specimens were divided into 25 pools, with 8 in each pool. The donors in each pool were both condition (with or without AUD) and sex balanced. For each pool, around 20 mg tissue from each donor specimen was collected and combined into a sterilized 2 ml Dounce homogenizer. 2 ml chilled NP40 lysis buffer (10 mM Tris-HCl, pH 7.5, 10 mM NaCl, 3 mM MgCl2, 0.1% Nonidet P40 Substitute, 1mM DTT, 1 U/l RNase inhibitor) was added to the Dounce homogenizer before the tissues were thawed. The tissues were homogenized 15x using pestle A, and 10x using pestle B, and were transferred into a centrifuge tube to incubate for 2 minutes on ice. After that, 2 ml wash buffer containing PBS, 1% BSA and 1 U/l RNase inhibitor was added and mixed well. The lysed tissue was centrifuged at 500 rcf for 5 minutes at 4C, then washed twice more with wash buffer and filtered through 70 m and then 40 m cell strainer separately. The pellet was resuspended in 2 ml wash buffer and mixed with 3.6 ml Sucrose Cushion Buffer I (nuclei PURE prep isolation kit, Sigma) containing 1 U/l RNase inhibitor. 2 ml Sucrose Cushion Buffer I with 1 U/l RNase inhibitor was added into one 15 ml Beckman Coulter centrifuge tube.  After that, the 5.6 ml nuclei suspensions were gently added to the top of Sucrose Cushion Buffer I without mixing, and followed by centrifuging at 13,000 x rpm (30,000 rcf) (Beckman Coulter ultracentrifuge) with rotor SW40Ti for 45 minutes at 4C. The purified nuclei pellet was washed by centrifuging at 300 rcf for 5 min at 4C with wash buffer, and the washed nuclei pellets was resuspended in wash buffer to target ~ 1000 nuclei/l.   10X Single-cell Multiome Library Preparation and Sequencing  Paired ATAC and gene expression libraries were generated following the Chromium Next GEM Single Cell Multiome ATAC + Gene Expression User Guide CG000338_RevB (10X Genomics, Inc). In brief, the isolated nuclei from a pool of samples were first incubated in a transposition mix. The single nuclei master mixture containing tagmented single nuclei suspension was loaded into two well of a Next GEM Chip J, along with the single cell multiome gel beads and partition oil. The chip was then loaded to the Chromium X Controller for GEM generation and barcoding. Barcoded transposed DNA and cDNA were amplified after GEMs being released. At each step, the quality of cDNA, ATAC library and cDNA library was examined by Bioanalyzer 2000. The final single indexed ATAC libraries and the dual indexed gene expression libraries were sequenced on an Illumina Novaseq 6000, with index reads of 10 bp + 24 bp, and 100 bp paired-end reads. The libraries were sequenced twice (2 technical replicates for each BioSample). Cell Ranger ARC Analysis  Cell Ranger ARC (cellranger-arc-2.0.0, http://support.10xgenomics.com/) was utilized to process the raw sequence data derived from the single-cell multiome libraries. Both the ATAC and gene expression FASTQ files were processed with the cellranger-arc count algorithm. The reference refdata-cellranger-arc-GRCh38-2020-A-2.0.0 (10x Genomics) was used. The filtered Demultiplexing: Cells from each of the sequencing pools were demultiplexed back into their samples of origin using the tool Demuxlet with default parameters. The data from the two technical replicates were then combined for the further analyses