Human trustain fcx fc blocking solution

Antibody staining of cell surface proteins was performed according to the Totalseq-A protocol (https://www.biolegend.com/en-us/protocols/totalseq-a-antibodies-and-cell-hashing-with-10x-single-cell-3-reagent-kit-v3-3-1-protocol) with modifications as follows.
A pooled suspension containing 2×10⁶ cells from 20 subjects at a time (~100,000 per subject) was centrifuged (300G, 5 min, 4°C) and resuspended in 100 µL Cell Staining Buffer (BioLegend, Cat. 420201) and incubated (10 min, 4°C) with 10 µL Human TruStain FcX™ Fc Blocking Solution (BioLegend, Cat. 422301). Cells suspensions were then stained (30 min, 4°C) with 100 µL TotalSeq antibody cocktail for feature barcoding of cell surface proteins (Supplementary Table 2) and divided into two 105 µL aliquots. Each aliquot was washed 3 times by resuspending in 15 mL Cell Staining Buffer and centrifuging (300G, 5 min, 4°C). Aliquots of washed cells were then resuspended in 150 µL 10% FBS in PBS to obtain a concentration of 1×10⁶ cells/mL, recombined, and filtered again with a 40 µm Flowmi Cell Strainer. Cell viability was measured with 10 µL of filtered cells by adding 10 µL 0.4% Trypan Blue and manually counting with a hemocytometer.
Cell density was adjusted to 2,500 cells/µL and run on the Chromium Controller (10X Genomics) using the Single Cell 3’ v3.1 Assay (10X Genomics) with a target of 50,000 cells per reaction.

Antibody staining of cell surface proteins was performed according to the Totalseq-A protocol (https://www.biolegend.com/en-us/protocols/totalseq-a-antibodies-and-cell-hashing-with-10x-single-cell-3-reagent-kit-v3-3-1-protocol) with modifications as follows. A pooled suspension containing 100,000 cells from at most 20 subjects at a time was centrifuged (300G, 5 min, 4°C) and resuspended in 100 µL Cell Staining Buffer (BioLegend, Cat. 420201) and incubated (10 min, 4°C) with 10 µL Human TruStain FcX™ Fc Blocking Solution (BioLegend, Cat. 422301). Cells suspensions were then stained (30 min, 4°C) with 100 µL TotalSeq antibody cocktail (Supplementary Table 2) and divided into two 105 µL aliquots. Each aliquot was washed 3 times by resuspending in 15 mL Cell Staining Buffer and centrifuging (300G, 5 min, 4°C). Washed cells were then resuspended in 150 µL 10% FBS in PBS, recombined, and filtered again with a 40 µm Flowmi Cell Strainer. Cell viability was measured with 10 µL of filtered cells by adding 10 µL 0.4% Trypan Blue and manual counting with a hemocytometer. Cell density was adjusted to 2,500 cells/µL and run on the Chromium Controller (10X Genomics) using the Single Cell 3’ v3.1 Assay (10X Genomics) with a target of 50,000 cells per reaction.