Abstract
Single-cell omics provide insight into cellular heterogeneity and function. Recent technological advances have accelerated single-cell analyses, but workflows remain expensive and complex. We present a method enabling simultaneous, ultra-high throughput single-cell barcoding of millions of cells for targeted analysis of proteins and RNAs. Quantum barcoding (QBC) avoids isolation of single cells by building cell-specific oligo barcodes dynamically within each cell. With minimal instrumentation (four 96-well plates and a multichannel pipette), cell-specific codes are added to each tagged molecule within cells through sequential rounds of classical split-pool synthesis. Here we show the utility of this technology in mouse and human model systems for as many as 50 antibodies to targeted proteins and, separately, >70 targeted RNA regions. We demonstrate that this method can be applied to multi-modal protein and RNA analyses. It can be scaled by expansion of the split-pool process and effectively renders sequencing instruments as versatile multi-parameter flow cytometers.
Highlights
Single-cell omics provide insight into cellular heterogeneity and function
Fluorescence-based flow cytometry enables the highest cell throughput (20–30,000 cells per second) of any technology far but is limited by the number of simultaneous parameters that can be determined per cell (10–15 by accomplished groups) with up to 28 possible reported in public conferences[5,6]
InsRT-Quantum barcoding (QBC) targeting 40 regions on 14 mRNA transcripts was applied to K562 cells, a chronic myeloid leukemia (CML) cell line with known fusion gene, BCR-ABL64–66
Summary
We employ the classic split-pool[37,45,46] process for assembling cell-specific barcodes on molecular targets, protein or RNA (Fig. 1a–e). A universal common oligonucleotide sequence is conjugated to antibodies. This common oligonucleotide anneals to a second oligonucleotide with a 9 bp unique barcode identifier, termed Assayable Hybridization Code Adapter (AHCA). The AHCA contains a region, termed the anchor, complementary to the Splint oligonucleotide. For RNA, (Fig. 1b) fixed and permeabilized cells are in situ reverse transcribed with gene specific primers that contain the same anchor sequence. The subcodes contain flanking regions designed to anneal the SC to a region on the Splint (Fig. 1d). See Supplementary Data 1–11 for oligo and primer sequences
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