Journal
BIOTECHNOLOGY JOURNAL
Volume 17, Issue 6, Pages -Publisher
WILEY-V C H VERLAG GMBH
DOI: 10.1002/biot.202100213
Keywords
antibody conjugation; CITE-seq; single-cell RNA-seq
Funding
- HiLIFE
- Biocenter Finland
- Finnish Foundation for Pediatric Research
- University of Helsinki's research grants
- Laboratoriolaaketieteen tukisaatio
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This study presents a simple adjustable oligonucleotide-antibody conjugation method that improves detection of weak protein targets in single-cell RNA sequencing (scRNA-seq) by optimizing oligonucleotide labeling. The method also reduces sequencing costs and is compatible with fixation and freeze storage protocols. The use of this method allows for flexibility and sample pooling in scRNA-seq multimodal experiments.
Background and Aim Single-cell RNA sequencing (scRNA-seq) is a powerful method utilising transcriptomic data for detailed characterisation of heterogeneous cell populations. The use of oligonucleotide-labelled antibodies for targeted proteomics addresses the shortcomings of the scRNA-seq-only based approach by improving detection of low expressing targets. However, optimisation of large antibody panels is challenging and depends on the availability of co-functioning oligonucleotide-labelled antibodies. Main Methods and Results We present here a simple adjustable oligonucleotide-antibody conjugation method which enables a desired level of oligo-conjugation per antibody. The mean labelling in the produced antibody batches varied from 1 to 6 oligos per antibody. In the scRNA-seq multimodal experiment, the highest sensitivity was seen with moderate antibody labelling as the high activation and/or labelling was detrimental to antibody performance. The conjugates were also tested for compatibility with the fixation and freeze storage protocols. The oligo-antibody signal was stable in fixed cells indicating the feasibility of a stain, fix, store, and analyse later type of workflow for multimodal scRNA-seq. Conclusions and Implications Optimised oligo-labelling will improve detection of weak protein targets in scRNA-seq multimodal experiments and reduce sequencing costs due to a more balanced amplification of different antibody signals in CITE-seq libraries. Furthermore, the use of a pre-stain, fix, run later protocol will allow for flexibility, facilitate sample pooling, and ease logistics in scRNA-seq multimodal experiments.
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