Multiome: RNA + ATAC of human pancreas (RNA modality): nPOD 6375
Roughly 40 mg ground tissue was resuspended. Nuclei were resuspended in 400 l of sort buffer, 1 U l1 RNasin and stained with 7-aminoactinomycin D. A total of 120,000 nuclei were sorted using an SH800 sorter into 87.5 l of collection buffer. Supernatant was gently removed, and ~10 l were left behind to increase nuclei recovery. A total of 650 l of wash buffer, 1% fatty acid-free BSA, 1 mM DTT , 1x protease inhibitors (Thermo Fisher Scientific, PIA32965), 1 U l1 RNasin (Promega, N2515) in molecular biology-grade water) was added with minimal disturbance of the pellet, and samples were centrifuged again with a swinging-bucket centrifuge (500  g, 5 min, 4 C; Eppendorf, 5920 R). Supernatant was gently removed without disturbing the pellet, leaving ~23 l behind. Approximately 710 l of 1x Nuclei Buffer (10x Genomics) were added, and nuclei were gently resuspended. Nuclei were counted using a hemocytometer, and 18,300 nuclei were used as input for tagmentation. Single-cell multiome ATAC and gene expression libraries were generated following manufacturer instructions (Chromium Next GEM Single Cell Multiome ATAC + Gene Expression Reagent Bundle, 10x Genomics, 1000283; Chromium Next GEM Chip J Single Cell Kit, 10x Genomics, 1000234; Dual Index Kit TT Set A, 10x Genomics, 1000215; Single Index Kit N Set A, 10x Genomics, 1000212) with the following PCR cycles: 7 cycles for pre-amplification, 8 cycles for ATAC index PCR, 7 cycles for complementary DNA (cDNA) amplification, and 12 cycles for RNA index PCR. Final libraries were quantified using a Qubit fluorometer (Life Technologies), and the size distribution was checked using a TapeStation (High Sensitivity D1000, Agilent). Libraries were sequenced on NovaSeq 6000 sequencers (Illumina) with the following read lengths (Read1 + Index1 + Index2 + Read2): ATAC (NovaSeq 6000), 50 + 8 + 16 + 50; RNA ( NovaSeq 6000), 28 + 10 + 10 + 91