Extended Data Fig. 3: Combinatorial indexing for single-cell MulTI-Tag.

a) Schematic describing single cell MulTI-Tag species mixing experiments. Human K562 cells (red) and mouse NIH3T3 cells (blue) were mixed and profiled in bulk, then cells were dispensed into nanowells on a Takara ICELL8 microfluidic device for combinatorial barcoding via amplification. b) Barnyard plots describing the number of unique fragments exclusively mapping to the hg19 genome build (X-axis) vs. mm10 (Y-axis) in all cells with greater than 100 unique reads for each of the denoted experiments. Points are colored by the cell identity as human (red; > 90% of unique reads mapping to hg19), mouse (blue; >90% mapping to mm10), or mixed (magenta; < 90% mapping to either), and collision rate, defined as the percentage of cells classified as ‘mixed’, is denoted for each experiment. c) Violin plots describing distributions of unique reads per cell in K562 cells (left), H1 cells (center), or the K562-H1 cell mixed population (right). Median values for total unique reads (black), H3K27me3 unique reads (red), or H3K36me3 unique reads (teal) are displayed at the top of each violin. Number of cells described is displayed at top of each cell type group. d) Violin plot describing distribution of fraction of on-target reads in peaks, defined as the percentage of reads corresponding to the same target for which the ENCODE ChIP-seq peak was called, in H3K27me3 (red) and H3K36me3 (teal) peaks from single cell MulTI-Tag in H1 cells (left) and K562 cells (right). Number of peaks is displayed above each violin. e) Violin plots describing Fraction of Reads in Peaks (FRiP) score in ENCODE ChIP-seq peaks for H3K27me3 (red) or H3K36me3 (teal) data from single cell CUT&Tag (white) or sequential single cell MulTI-Tag (grey). Number of cells described and number of peaks used is displayed below each violin. f) Jittered scatterplot describing the number of counts mapping to each single cell within each of the indicated genes in single cell CUT&Tag²⁹ (black) vs. single cell MulTI-Tag (grey). The percentage of cells with non-zero counts for each locus and assay are denoted at the bottom. g) Table describing comparative metrics for MulTI-Tag (this study) in comparison with scMulti-CUT&Tag²¹, scCUT&Tag^28,29, and scChIP-seq³².