Recovery and analysis of transcriptome subsets from pooled single-cell RNA-seq libraries

Kent A. Riemondy, Monica Ransom, Christopher Alderman, Austin E. Gillen, Rui Fu, Jessica Finlay-Schultz, Gregory D. Kirkpatrick, Jorge Di Paola, Peter Kabos, Carol A. Sartorius, Jay R. Hesselberth

Research output: Contribution to journalArticle

2 Scopus citations

Abstract

Single-cell RNA sequencing (scRNA-seq) methods generate sparse gene expression profiles for thousands of single cells in a single experiment. The information in these profiles is sufficient to classify cell types by distinct expression patterns but the high complexity of scRNA-seq libraries often prevents full characterization of transcriptomes from individual cells. To extract more focused gene expression information from scRNA-seq libraries, we developed a strategy to physically recover the DNA molecules comprising transcriptome subsets, enabling deeper interrogation of the isolated molecules by another round of DNA sequencing. We applied the method in cell-centric and gene-centric modes to isolate cDNA fragments from scRNA-seq libraries. First, we resampled the transcriptomes of rare, single megakaryocytes from a complex mixture of lymphocytes and analyzed them in a second round of DNA sequencing, yielding up to 20-fold greater sequencing depth per cell and increasing the number of genes detected per cell from a median of 1313 to 2002. We similarly isolated mRNAs from targeted T cells to improve the reconstruction of their VDJ-rearranged immune receptor mRNAs. Second, we isolated CD3D mRNA fragments expressed across cells in a scRNA-seq library prepared from a clonal T cell line, increasing the number of cells with detected CD3D expression from 59.7% to 100%. Transcriptome resampling is a general approach to recover targeted gene expression information from single-cell RNA sequencing libraries that enhances the utility of these costly experiments, and may be applicable to the targeted recovery of molecules from other single-cell assays.

Original languageEnglish
Article numbere20
JournalNucleic acids research
Volume47
Issue number4
DOIs
StatePublished - Feb 28 2019
Externally publishedYes

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    Riemondy, K. A., Ransom, M., Alderman, C., Gillen, A. E., Fu, R., Finlay-Schultz, J., Kirkpatrick, G. D., Di Paola, J., Kabos, P., Sartorius, C. A., & Hesselberth, J. R. (2019). Recovery and analysis of transcriptome subsets from pooled single-cell RNA-seq libraries. Nucleic acids research, 47(4), [e20]. https://doi.org/10.1093/nar/gky1204