Fig. 1
From: Immuno-detection by sequencing enables large-scale high-dimensional phenotyping in cells
Immuno-detection by sequencing (ID-seq) technology development. a Concept of the ID-seq technology. First, pool DNA-tagged antibodies. Second, perform multiplexed immunostaining on fixed cell populations, and release DNA tags. Third, barcode the released DNA tags through a two-step PCR protocol. Finally, sequence the barcoded DNA tags via next-generation sequencing (NGS) and count barcodes. b Signals from singleplex epitope detection (via an immuno-PCR measurement) were compared with multiplexed epitope detection using ID-seq. The histogram summarises correlations between 17 immuno-PCR and corresponding ID-seq measurements. Insert panel illustrates an example from the actin antibody showing signal mean and s.d. (n = 4). Underlying data for all 17 antibodies can be found in Supplementary Fig. 4. c Scatterplot indicates the high reproducibility of PCR-based ID-seq library preparation (r = Pearson correlation). Libraries from the same released material were prepared on separate occasions and analysed in different sequencing runs. d The scatterplot shows the counts (mean) and coefficient of variation from 69 antibody–DNA conjugates (n = 14 biological replicates). Dashed line indicates 20% variation. e Volcano plot shows the effect (estimate) and significance (−log10 pval) of AG1478 treatment (n = 6), based on the model analysis of ID-seq counts (Supplementary Note 3). Significance (−log10 pval) determines node size. Red nodes show significantly increased (ANOVA, p < 0.01) and blue nodes show significantly decreased (ANOVA, p < 0.01) (phosphor-)protein levels. f Pathway overview of ID-seq measurements after 48 h of AG1478 treatment. Colour indicates the effect size and node size represents the significance of effect (−log10 pval). Light grey nodes without border indicate not measured (nm) proteins (see Supplementary Supplementary Fig. 12 for (phospho-)protein identities)
