Extended Data Fig. 2: Species mixing QC analysis (Human/Mouse).

a, (Upper) Transcriptomic UMAP of human and mouse cells, distinguished by transcript alignment. (Lower) ADT staining of mouse and human cells overlaid on the transcriptomic UMAP for 100k loading experiment. Pool barcodes per antibody were merged (that is CD29_h_merged-1 = CD29_h_barcode-1 + ….+CD29_h_barcode-5, where the latter number represents the pool number). Species classification was transcriptomically determined by a > 95% cutoff based on normalized counts specific to either species. Cells which did not meet the threshold were classified as Multiplets. Overlaid normalized ADT counts shows human and mouse antibody staining. b, Scatterplot showing within species multiplets (shown on double-positive axes) across batches when loading 100k cells. Resolution of cell types with a single batch barcode and annotation of Multiplets (positive for both pool barcodes, such as CD29h_barcode1 and CD29h_barcode2 positive or CD29m_barcode1 and CD29m_barcode2). c, Scatterplots for species mixing 20k (left) and 100k (right) loading experiments colored by pool showing pool specific staining level. Resolved hCD29 or mCD29 on the axes refers to normalized antibody counts after resolution into single cells. If a droplet contained a mixture of hCD29 from pool 1 and pool 2, the droplet was resolved as two cells with the pool-normalized counts. d, Scatter plots of SCITO-seq normalized counts from 2 × 10⁴ loading of species mixing to determine cross pool or within pool background level. e, Scatter plots of directly-conjugated hCD29 and mCD29 antibody-based normalized counts from 2 × 10⁴ loading of species mixing to show cross pool or within pool background level using direct conjugation. Hg19, mm10, and Multiplet define cell populations based on respective transcriptomic alignment. Direct conjugates provide a baseline for noise in the SCITO-seq system.