title: GSM7100395: Mux24_HTO; Mus musculus; RNA-Seq
library_construction_protocol: Nuclei were isolated from frozen mouse inguinal white adipose tissue samples for 10X snRNA-seq as previously described, with the following modifications to integrate hash multiplexing and FANS-assisted nuclear quality thresholding and sample pooling. Briefly, 300 mg of flash-frozen adipose samples were held on dry ice until immediately before nuclei isolation, and all sample handling steps were performed on ice. Each sample was placed into a gentleMACS C tube (Miltenyi Biotec) with 2 mL freshly prepared TST buffer (0.03% Tween 20 (Bio-Rad), 0.01% Molecular Grade BSA (New England Biolabs), 146 mM NaCl (ThermoFisher Scientific), 1 mM CaCl2 (VWR International), 21 mM MgCl2 (Sigma Aldrich), and 10 mM Tris-Hcl pH 7.5 (ThermoFisher Scientific) in Ultrapure water (ThermoFisher Scientific)) with 0.2 U/μL of Protector RNase Inhibitor (Sigma Aldrich). gentleMACS C tubes were then placed on the gentleMACS Dissociator (Miltenyi Biotec) and tissue was dissociated by running the program "mr_adipose_01" three times, and then incubated on ice for 10 minutes. Lysate was passed through a 40 μm nylon filter (CellTreat) and collected into a 50 mL conical tube (Corning). Filter was rinsed with 3 mL of freshly prepared ST buffer (146 mM NaCl, 1 mM CaCl2, 21 mM MgCl2; 10 mM Tris-Hcl pH 7.5) with 0.2 U/μL RNase Inhibitor, and collected into the same tube. Flow-through was passed through a 20 μm pre-separation filter (Miltenyi Biotec) set on top of a 5 mL FACS tube (Corning) and collected into the same tube. Suspension was centrifuged in a swinging-bucket centrifuge (Eppendorf) at 500 × g for 5 minutes at 4°C with brake set to low. Following centrifugation, supernatant was removed and 5 mL of PBS pH 7.4 (ThermoFisher Scientific) with 0.02% BSA and 0.2 U/μL RNase Inhibitor was added without resuspending the nuclear pellet. Sample was centrifuged again at 500 × g for 5 minutes at 4°C with brake set to low. Following centrifugation, supernatant was removed, and the nuclear pellet was resuspended in 1 mL PBS-0.02% BSA with 0.2 U/μL RNase Inhibitor. Each sample was split into two 500 μL aliquots and transferred to new 5 mL FACS tubes for subsequent hashing. Each aliquot of resuspended nuclei was stained with NucBlue (Thermofisher Scientific), labeled with 1 μg of a unique TotalSeq anti-Nuclear Pore Complex Proteins Hashtag Antibody (Biolegend), and then incubated on ice for 30 minutes. Suspension was centrifuged at 500 × g for 5 minutes at 4°C with brake set to low. Following centrifugation, 450 μL of supernatant was removed and the nuclear pellet was resuspended in 450 μL PBS-0.02% BSA with 0.2 U/μL RNase Inhibitor. For nuclear quality thresholding, fluorescence-activated nuclear sorting (FANS) was implemented to collect 4,000-4,300 nuclei from hashtagged aliquots directly into a shared well of a 96-well PCR plate (Thermo Scientific) containing 24.6 μL of 10X RT Reagent B with 1U/uL RNase Inhibitor on a Beckman Coulter MoFlo AstriosEQ fitted with a 70 μm nozzle. High-quality nuclei were selected by initial gating at 360 nm with laser filter 405-448/59 followed by SSC-H and FSC-H to remove doublets and unlysed cells. Once all sample aliquots were FANS-sorted, the pool of 43,000 nuclei was loaded on the 10x Chromium controller (10x Genomics) according to the manufacturer's protocol. cDNA and gene expression libraries were generated according to the manufacturer's instructions (10x Genomics). Libraries of hashtag oligo fractions were generated according to the manufacturer's instructions (Biolegend). cDNA and gene expression library fragment sizes were assessed with a DNA High Sensitivity Bioanalyzer Chip (Agilent). cDNA and gene expression libraries were quantified using the Qubit dsDNA High Sensitivity assay kit (ThermoFisher Scientific). Gene expression libraries were multiplexed and sequenced on the Nextseq 500 (Illumina) using a 75-cycle kit and the following read structure: Read 1: 28 cycles, Read 2: 55 cycles, Index Read 1: 8 cycles. Raw sequencing reads were demultiplexed to FASTQ format files using bcl2fastq (Illumina; version 2.20.0). Digital expression matrices were generated from the FASTQ files using Cell Ranger (version 6.1.2) with the option to include intronic reads (--include-introns). Reads were aligned against the GRCm38 mouse genome assembly and gene counts were obtained, per-droplet, by summarizing exonic and intronic UMIs that overlapped with the GENCODE mouse annotation (release 24) for each gene symbol.