Ultra-high throughput single-cell analysis of proteins and RNAs by split-pool synthesis

Single-cell omics provide insight into cellular heterogeneity and function. Recent technological advances have accelerated single-cell analyses, but workflows remain expensive and complex. Researchers at Roche Sequencing Solutions 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 the researchers 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. They 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.

Methodology and Schematics of Single-cell Barcoding by Split Pool


a Method for single-cell QBC antibody coding. Antibodies are conjugated to a linker oligonucleotide. An AHCA barcode oligo is annealed to the linker oligo prior to staining the cells with the antibody-conjugate annealed to AHCA oligo complex. This AHCA oligo also contains an “anchor” sequence for later annealing of the Splint. Cells are stained with pools of antibodies prepared this way. b Method for single-cell QBC mRNA coding by reverse transcription. A panel of gene specific primers is added to fixed cells and reverse transcription is initiated. Splint is added via annealing to the complementary anchor region on the reverse transcription initiator. c Split-pool steps for quantum barcoding. Step 1. Cells are randomly split into 60 wells of a 96-well microtiter plate each of which contains one of 60 unique short oligonucleotides. Step 2. The SC subcode anneals to the Splint and is ligated to the adjacent sequence. Cells are washed of free SC. Step 3. Cells are pooled and allowed to go through the split-pool process with successive sets of SC oligos. The process is repeated a total of four times. d Schematic description of general QBC method. The respective reverse transcribed or antibody bound probes from 1a-b in cells are treated with the Splint sequence. The Splint sequence binds to the cognate oligo in the cell. Excess Splint is washed away. The cells are subjected to the split-pool process outlined in Fig. 1c. The codes are annealed on the Splint and ligated in place to link the structure together. To avoid mismatches at the barcode region, the Splint oligo has carbon spacers (C-C) to allow different barcode sequences to anneal. At the end of the split-pool process, the cells are lysed and DNA prepared for PCR. e Final structure of libraries to be sequenced. Post PCR, the final structure of the material(s) are shown representing either the presence of antibodies or a given targeted mRNA. At the top of the panel are the extracted code sequences removed with the parsing program.

O’Huallachain M et al. (2020) Ultra-high Throughput Single-Cell Analysis of Proteins and RNAs by Split-Pool Synthesis. Commun Biol 3(1):213. [article]

Leave a Reply

Your email address will not be published. Required fields are marked *


Time limit is exhausted. Please reload CAPTCHA.