Pluto Bioinformatics

GSE150284: CoolMPS for robust sequencing of single-nuclear RNAs captured by droplet-based method

Bulk RNA sequencing

Massively-parallel single-cell and single-nucleus RNA sequencing (scRNA-seq, snRNA-seq) requires extensive sequencing to achieve proper per-cell coverage, making sequencing resources and availability of sequencers critical factors for conducting deep transcriptional profiling. CoolMPS is a novel sequencing-by-synthesis approach relying on nucleotide labeling by re-usable antibodies, but whether it is applicable to scRNA-seq, has not been tested. Here, we use a low-cost and off-the-shelf protocol to chemically convert libraries generated with the widely-used Chromium 10X technology to be sequenceable with CoolMPS. To assess the quality and performance of converted libraries, we generated a snRNA-seq dataset from the hippocampus of young and old mice. Native libraries were sequenced on an Illumina Novaseq and, after conversion, on a DNBSEQ-400RS. CoolMPS-derived results were in very high agreement with the Illumina dataset, and we demonstrate how technical variance between both technologies is significantly lower when compared to biological variance. Indeed, CoolMPS-derived data faithfully replicated key characteristics of the native library dataset, including correct estimation of ambient RNA-contamination, detection of captured cells, cell clustering, spatial marker gene expression, inter- and intra-replicate differences and gene expression changes. In conclusion, our results show that CoolMPS provides a viable alternative to standard sequencing of RNA from droplet-based libraries. SOURCE: Tobias Fehlmann Saarland University

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