@article {Riemondy408740, author = {Kent A. Riemondy and Monica Ransom and Christopher Alderman and Austin E. Gillen and Rui Fu and Jessica Finlay-Schultz and Gregory Kirkpatrick and Jorge Paola Di and Peter Kabos and Carol A. Sartorius and Jay R. Hesselberth}, title = {Recovery and analysis of transcriptome subsets from pooled single-cell RNA-seq libraries}, elocation-id = {408740}, year = {2018}, doi = {10.1101/408740}, publisher = {Cold Spring Harbor Laboratory}, 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 1,313 to 2,002. 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.}, URL = {https://www.biorxiv.org/content/early/2018/09/05/408740}, eprint = {https://www.biorxiv.org/content/early/2018/09/05/408740.full.pdf}, journal = {bioRxiv} }