Extended Data Fig. 7: Scalability experiment and QC analysis (60- and 165-plex).
From: SCITO-seq: single-cell combinatorial indexed cytometry sequencing
a, Design of splint oligo with Totalseq-C compatible system. The splint oligos (FBC RC (reverse complement) + Well + Ab BC (5 + 5,10 bp) + Read2 + Totalseq-C Barcode RC) are hybridized to the barcode region of the Totalseq-C oligo conjugated antibodies (right dotted lines around the blue region). Splint oligo (1 μM) was incubated with antibody for 15 min for hybridization (same workflow as conventional SCITO-seq). The well and the antibody barcode sequences are encoded in orange and blue above. b, Ridgeplots of 60-plex experiment showing the specificity of the pool specific antibodies. The normalized expression values of the sum of counts for all 60 antibodies per pool. Individual plots contain batch specific normalized expression values to show the signal-to-noise distribution of the expected specificity. c, Ridgeplots of 165-plex experiment showing the specificity of the pool specific antibodies. The normalized expression values of the sum of all 165 antibodies per pool. Individual plots contain batch specific normalized expression values to show signal to noise distribution of the expected specificity. d, Barplots of 165-plex experiment showing low UMI counts of an example Isotype control ADT counts (Rat IgG) for all 10 pools (top 1-5 batches, bottom 6-10 batches). The percentage of cells (y-axis) that express the ADT less than 2 UMI or over is calculated. Background noise across batches shows less than 2 UMI counts in ~92% of the cells. e, Overlay density histograms of the example CD8a vs Isotype control Ab (Rat IgG) to assess the ‘noise’ level for all 10 pools (top 1-5 batches, bottom 6-10 batches) in 165-plex data. X-axis for log1p(raw counts) transformed values and y-axis for density. f, Overlay density histograms of the example antibodies aggregated over all 10 pools (CD3, CD11c, CD45, CD127, CD8a, CD4, CD19) vs Isotype control Ab (Rat IgG) to assess the ‘noise’ level in 165-plex data. X-axis for log1p(raw counts) transformed values and y-axis for density.
