Ultra-high throughput single-cell RNA sequencing by combinatorial fluidic indexing

Supplementary Website

Cell atlas projects and high-throughput perturbation screens require single-cell sequencing at a scale that is challenging with current technology. To enable cost-effective single-cell sequencing for millions of individual cells, we developed ‘single-cell combinatorial fluidic indexing’ (scifi). The scifi-RNA-seq assay combines one-step combinatorial preindexing of entire transcriptomes inside permeabilized cells with subsequent single-cell RNA-seq using microfluidics. Preindexing allows us to load several cells per droplet and computationally demultiplex their individual expression profiles. Thereby, scifi-RNA-seq massively increases the throughput of droplet-based single-cell RNA-seq, and provides a straightforward way of multiplexing thousands of samples in a single experiment. Compared with multiround combinatorial indexing, scifi-RNA-seq provides an easy and efficient workflow. Compared to cell hashing methods, which flag and discard droplets containing more than one cell, scifi-RNA-seq resolves and retains individual transcriptomes from overloaded droplets. We benchmarked scifi-RNA-seq on various human and mouse cell lines, validated it for primary human T cells and applied it in a highly multiplexed CRISPR screen with single-cell transcriptome readout of T cell receptor activation.


scifi-RNA-seq overview

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Pre-indexing single-cell transcriptomes

We use barcoded reverse transcription primers to index transcriptomes of permeabilized cells with a round1 barcode shared by all cells in the same well.

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Microfluidics with droplet overloading

We randomly mix pre-indexed cells and overload the microfluidic droplet generator up to 100-fold. Multiple cells in the same droplet receive the same microfluidic round2 barcode.

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Deconvolution of single-cell transcriptomes

Based on the combination of round1 and round2 barcodes, we obtain pure single-cell transcriptomes by deconvolution of sequencing reads.

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Ultra-high throughput scRNA-seq with sample multiplexing

scifi-RNA-seq enables ultra-high throughput scRNA-seq with multiplexing for thousands of samples, for instance for large perturbation screens at the single-cell level.

News and updates

Date Description
2020/09/15

scifi-RNA-seq protocol design

Our method is based on an innovative thermoligation step to connect round1 and round2 barcodes inside the emulsion droplets.
Therefore, scifi barcoding can easily be extended to other single-cell assays and multi-omics protocols
Any particular application in mind? Contact us: scifi-RNA-seq@medical-epigenomics.org

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2019/12/18

scifi-RNA-seq pre-print published on bioRxiv

We are excited to share our pre-print article on scifi-RNA-seq (DOI: 10.1101/2019.12.17.879304).

Protocols and reagents

Updated protocols and oligonucleotide sequences for scifi-RNA-seq.

Date Description
2020/09/15

scifi-RNA-seq step-by-step protocol (Version: 2021/01/25)

Detailed step-by-step description of the scifi-RNA-seq method

The protocol contains a detailed listing of all required reagents and oligonucleotide sequences.
For each of the major steps, we present and discuss the expected results.

Download scifi-RNA-seq protocol (PDF)

Download table with oligonucleotide sequences (Excel)

Data and software

Links to raw data archives and related resources.

Name Description and link
Raw and processed data GEO accession: GSE168620.
Data processing pipeline Git repository at Github: https://github.com/epigen/scifiRNA-seq
Code for scifi-RNA-seq paper Git repository at Github: https://github.com/epigen/scifiRNA-seq_publication

Citation

If you use this method in your research, please cite:

Paul Datlinger*, André F Rendeiro*, Thorina Boenke, Martin Senekowitsch, Thomas Krausgruber, Daniele Barreca, Christoph Bock. Ultra-high throughput single-cell RNA sequencing by combinatorial fluidic indexing. Nature Methods 18, 635–642 (2021). doi.org/10.1038/s41592-021-01153-z