Totalseq anti human hashtag antibody

Single cells were washed with dPBS + Y-27632, resuspended in Advanced Dulbecco’s modified Eagle medium/F12 + 1% bovine serum albumin + Y-27632, and then stained with AnnexinV-Allophycocyanin (APC) (1:100) and 1 TotalSeq Anti-Human Hashtag Antibody (1:100, Biolegend, San Diego, CA) per region to track all 6 regions with a single library preparation.¹¹ (link) Cells were washed and resuspended in Advanced Dulbecco’s modified Eagle medium + 1% bovine serum albumin + Y-27632 for sorting on a Sony Cell Sorter SH800Z (Sony, Tokyo, Japan) to enrich for live single epithelial cells (Figure 18). There were 25,000 cells that passed Annexin V live/dead parameters and were fluorescence-activated cell sorter isolated for each region, then different cell hashing antibodies were added to cells from each of the 6 regions before pooling for library preparation. An additional live/dead analysis was performed after pooling and approximately 10,000 cells from the pooled population were loaded onto the Chromium Next GEM Single Cell 3’ GEM, Library and Gel Bead Kit v3.1 (PN-100012, 10x Genomics, Pleasanton, CA) for complementary DNA library preparation Sequencing was performed on an Illumina NextSeq 500 (Illumina, San Diego, CA).Figure 18

Fluorescence-activated cell sorter (FACS) strategy. FACS strategy for gating out cell fragments, likely doublets, and dead cells. APC, Allophycocyanin.