Immuno-Detection by sequencing (ID-seq) enables large-scale high-dimensional phenotyping in cells

Abstract

Cell-based small molecule screening is an effective strategy leading to new medicines. Scientists in the pharmaceutical industry as well as in academia have made tremendous progress in developing both large-scale and smaller-scale screening assays. However, an accessible and universal technology for measuring large numbers of molecular and cellular phenotypes in many samples in parallel is not available. Here, we present the Immuno-Detection by sequencing (ID-seq) technology that combines antibody-based protein detection and DNA-sequencing via DNA-tagged antibodies. We used ID-seq to simultaneously measure 84 (phospho-)proteins in hundreds of samples and screen the effects of ~300 kinase inhibitor probes on primary human epidermal stem cells to characterise the role of 225 kinases. Our work highlighted a previously unrecognized downregulation of mTOR signaling during differentiation and uncovered 13 kinases regulating epidermal renewal through distinct mechanisms.

Single-cell ID-seq identifies BMP signaling as a driver of a late stage epidermal differentiation program

“…We applied single-cell ID-seq to monitor the activity of signaling pathways and other cellular processes during epidermal differentiation using a panel of 70 antibody-DNA conjugates (Buggenum et al, 2017;Van Buggenum et al, 2016). These antibodies cover a broad range of cellular processes including cell cycle, DNA damage, epidermal self-renewal and differentiation, as well as the intracellular signalling status for the EGF, G-protein coupled receptors, calcium signalling, TNFα, TGFβ, Notch, WNT and BMP pathways (Buggenum et al, 2017 and supplemental table 1).…”

“…These antibodies cover a broad range of cellular processes including cell cycle, DNA damage, epidermal self-renewal and differentiation, as well as the intracellular signalling status for the EGF, G-protein coupled receptors, calcium signalling, TNFα, TGFβ, Notch, WNT and BMP pathways (Buggenum et al, 2017 and supplemental table 1). This panel includes 34 antibodies against phosphorylated epitopes and was previously validated and used to identify kinases involved in epidermal renewal (Buggenum et al, 2017). For 11 of the phosphorylated epitopes we were also able to measure the non-phosphorylated protein, allowing us to correct for total protein levels by calculating the phosphorylated to total protein ratio per cell.…”

“…For 11 of the phosphorylated epitopes we were also able to measure the non-phosphorylated protein, allowing us to correct for total protein levels by calculating the phosphorylated to total protein ratio per cell. Most of the targeted processes are measured using multiple (3-5) independent validated antibodies (Buggenum et al, 2017).…”

“…For the library preparation 3 PCR steps were performed to amplify the antibody barcodes and to add barcodes specific for the well and the plate of each cell. The barcoding occurred with the same sequences used in ID-seq (Buggenum et al, 2017). For the first PCR step 15 cycles were run after adding to each well a 4 ul reaction mix containing the Herculase II Fusion DNA Polymerase (Agilent), dNTPs, 5x Herculase buffer and 0.1µM amplification primers (Forward 5ʹ-CACGACGCTCTTCCGATCT-3ʹ, Reverse 5ʹ-TCGCTTATCTGTTGACTGAT-3ʹ).…”

“…Results scID-seq allows robust, reproducible and highly multiplexed protein detection in single cells. We recently described the Immuno-Detection by sequencing (ID-seq) technology to quantify >70 proteins and phospho-proteins in hundreds of cell populations simultaneously (Buggenum et al, 2017;Van Buggenum et al, 2016). In short, ID-seq entails immuno-staining with DNA-barcoded antibodies followed by quantification through high-throughput sequencing and barcode counting.…”