Human PBMCs Labeled With Extracellular Labeling Antibodies Using the TotalSeq™-C Human Universal Cocktail and Intracellular Antibodies Using a Custom Panel of Intracellular Labeling TotalSeq-C Human Antibodies From BioLegend

Human PBMCs, obtained by 10x Genomics from AllCells, were placed in culture and rested overnight. The following day, the cells were subjected to one of three conditions:

1. Rest: Cells continued resting in culture.
2. PMA/Ionomycin stimulation: Cells were stimulated for 6 hours with PMA and ionomycin in the presence of protein transport inhibitors, brefeldin A and monensin, using the eBioscience™ Cell Stimulation Cocktail (plus protein transport inhibitors, Cat# 00-4975-93).
3. LPS stimulation: Cells were stimulated for 6 hours with 5 ng/mL LPS (InvivoGen LPS-B5 Ultrapure, Cat# tlrl-pb5lps) alongside protein transport inhibitors brefeldin A and monensin, using the eBioscience™ Protein Transport Inhibitor Cocktail (Cat# 00-4980-03).

After stimulation, cells were harvested and any cells attached to the flask were recovered with accutase treatment. The cells were then cryopreserved.

To perform antibody (Ab) labeling with extracellular and intracellular antibodies, cryovials from the three conditions were thawed. Cells were either fixed for 1 hour at room temperature (condition: Rested, no Ab) or Ab labeled (Conditions: Rested, PMA/Iono, LPS) following Demonstrated Protocol Cell Surface & Intracellular Protein Labeling for GEM-X Flex Gene Expression (CG000781, Rev A). The extracellular antigens were labeled with the TotalSeq™-C Human Universal Cocktail, v1.0 (BioLegend, Cat#G900004) following the Cell Surface Protein Labeling Protocol section of the protocol, with the minor modification that the staining was done in a total of 50 μL (BioLegend recommendation for TotalSeq™ Universal Cocktails). Cells were washed using the 2-Wash option. Intracellular antigens were then labelled with 10 TotalSeq™-C Abs (CD20 cytoplasmic domain, Perforin, GranzymeA, GranzymeB, IL6, IL4, TNF, IL2, IL17a, IFNg) following the Intracellular Protein Labeling Protocol section of the protocol. After Ab labeling, cells were then fixed for 1 hour at room temperature following Fixation of Cells & Nuclei for GEM-X Flex Gene Expression Demonstrated Protocol (CG000782).

Gene Expression and Protein libraries were generated as described in the GEM-X Flex Gene Expression Reagent Kits for Multiplexed Samples with Feature Barcode technology for Protein using Barcode Oligo Capture User Guide (CG000789) and sequenced on an Illumina NovaSeq 6000 with approximately 18,000 paired end reads per cell for Gene Expression and 31,000 paired end reads/cell for Feature Barcode libraries.

Number of cells called by condition:

• Rested, no Ab: 17,588
• Rested, with Ab: 19,296
• PMA/Iono induced, with Ab: 15,319
• LPS induced, with Ab: 18,225

Paired-end, dual indexing libraries were sequenced using the following scheme:

• 28 cycles Read 1
• 10 cycles i7
• 10 cycles i5
• 90 cycles Read 2

This dataset is licensed under the Creative Commons Attribution 4.0 International (CC BY 4.0) license. 10x citation guidelines available here.