title: Pooled single cell Epitope library of human adult PBMCs
design: TEA-seq library preparation (Swanson et. al., 2021) was performed as with the addition of Cell Hashing to allow for sample multiplexing and limit well-to-well batch effects. In brief, samples were thawed and processed across three batches, with each batch containing a common PBMC control. Antibody staining for Cell Hashing and cell sorting was performed simultaneously on 2 ´ 106 cells from each sample. Each sample was incubated with a sample-specific barcoded TotalSeq-A antibody, anti-CD45 antibody, and anti-CD3 antibody, then pooled by T cell proportions previously determined by flow cytometry, targeting 800,000 T cells for each donor sample and 200,000 T cells for the control, and sorted on a BD FACSAria Fusion. T cells were sorted as live single CD45+CD3+ cells; 2 ´ 106 sorted T cells were then used for library preparation. A panel of 55 target-specific barcoded oligo-conjugated antibodies (BioLegend TotalSeq-A) was used for these studies. Individual ATAC, RNA, HTO, and ADT libraries were prepared, sequenced, and processed.