Totalseq c antibodies

Simultaneous evaluation of mRNA and cell surface expression from single cells was performed using feature barcoding (FB) technology from 10× Genomics, based on the CITE-seq technology (Stoeckius et al., 2017 (link)). Cell hashing (HTO) was used in conjunction with the 10× Genomics 5’V(D)J Feature Barcoding Kit to generate single-cell mRNA gene expression (GEX) and antibody-derived tag (ADT) libraries (Stoeckius et al., 2017 (link); Stoeckius et al., 2018 (link)). Briefly, PBMC from 12 samples were hashed using TotalSeq-C anti-human Hashtag antibodies and combined into two batches. In each batch, surface proteins were stained with a cocktail of 53 TotalSeq-C antibodies (BioLegend). Antibody concentrations were either predetermined by titration (Kotliarov et al., 2020 (link)) or used at a default concentration. 50,000 cells from each batch were loaded onto each of four wells of a Chromium chip, and GEX and ADT (HTO and FB) libraries were constructed following the manufacturer’s protocol. Libraries were pooled and quantitated using a MiSeq Nano v2 reagent cartridge. Final libraries were sequenced on the NovaSeq 6000, S4 reagent cartridge (2×100 bp) (Illumina).

Frozen PBMC samples were thawed quickly at 37 °C in a water bath. Warm RPMI1640 medium (20–30 ml) containing 10% FBS was added slowly to the cells before centrifuging at 300g for 5 min. This was followed by a wash in 5 ml RPMI1640-FBS. The PBMC pellet was collected, and the cell number and viability were determined using Trypan Blue. PBMCs from four different donors were then pooled together at equal numbers: 1.25 × 10⁵ PBMCs from each donor were combined with the other PBMCs to make up 5.0 × 10⁵ cells in total. The remaining cells were used for DNA extraction (Qiagen, 69504). The pooled PBMCs were resuspended in 25 µl of cell staining buffer (BioLegend, 420201) and blocked by incubation for 10 min on ice with 2.5 µl Human TruStain FcX block (BioLegend, 422301). The PBMC pool was then stained with TotalSeq-C antibodies (BioLegend, 99814) according to the manufacturer’s instructions. For a full list of TotalSeq-C antibodies, refer to ref. ^{23 (link)}. After incubating with 0.5 vials of TotalSeq-C for 30 min at 4 °C, PBMCs were washed three times by centrifugation at 500g for 5 min at 4 °C. PBMCs were counted again and processed immediately for 10x 5′ single cell capture (Chromium Next GEM Single Cell V(D)J Reagent Kit v1.1 with Feature Barcoding technology for cell Surface Protein-Rev D protocol). Two lanes of 25,000 cells were loaded per pool onto a 10x chip.

During primary staining, splenocytes were additionally labeled with TotalSeq-C antibodies (BioLegend): prediabetic VH125.NOD mouse 1 – TotalSeq-CO301 hashtag 1 with 5′– ACCCACCAGTAAGAC–3′ barcode, mouse 2 – TotalSeq-CO302 hashtag 2 with 5′– GGTCGAGAGCATTCA–3′ barcode, and mouse 3 – TotalSeq-CO303 hashtag 3 with 5′– CTTGCCGCATGTCAT–3′ barcode. Labelling with hashtag antibodies was performed on ice for 30 min. After sorting, high-affinity IBCs from three VH125.NOD mice were pooled for library construction using 10X Genomics protocols and Illumina sequencing.

Mice were euthanized by isoflurane overdose and cervical dislocation and perfused with 20 ml PBS through the left ventricle. eAT pads were collected, minced, and digested in 6 ml of 2-mg/mL type II collagenase (Worthington # LS004177) for 30 min at 37 °C. Digested eAT was then vortexed, filtered through 100 μm filters, lysed with ACK buffer, and filtered through 35 μm filters as previously described⁸¹ .
The AT stromal vascular fraction was prepped with anti-mouse Fc Block (BD Biosciences) at 1:200. Cells from each mouse were labeled with unique hashtag antibodies (1:200) (Biolegend TotalSeq-C) and anti-CD45 microbeads (10 μL/ sample) (Miltenyi # 130-052-301). Biological replicates were pooled and sorted on a Miltenyi AutoMACs using the “possel_s” option. CD45⁺ cells were then labeled for CITE-sequencing using TotalSeq-C antibodies (Biolegend) for cell surface markers to identify major cell types. All immunolabeling was completed at a 1:200 dilution for 20 min at 4 °C in the dark. More information regarding specific samples and antibody manufacturer/catalog numbers can be found in Supplementary Table 1. Cells were stained with 0.25 µg/mL DAPI for FACS sorting and DAPI⁻ viable cells were collected for downstream processing and sequencing.

After providing written informed consent, the patient sample was collected after progression on venetoclax treatment [46 ] (Human Research Ethics Committee approvals: Melbourne Health 2011.044, 2016.305, 2012.274; Peter MacCallum Cancer Centre 11/18; Walter and Eliza Hall Institute 05/04, 13/01). Blood was collected in EDTA tubes and processed within 2 h. Peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll-Paque Plus (#17144002, lot:10258101, GE Healthcare) density gradient centrifugation and cells were cryopreserved. PBMCs were thawed, rested for 2 h, and incubated with Fc Receptor blocking solution (Human TruStain FcX, Biolegend) for 10 min prior to staining with TotalSeq C antibodies (Biolegend) at 4 ° for 30 min. PBMCs were washed three times and stained with propidium iodide (PI, Sigma). Viable cells (PI negative) were flow sorted using the FACSAria (BD) and diluted to 1000 cells/μL.