High-throughput single-cell assays increasingly require special consideration in experimental design, sample multiplexing, batch effect removal, and data interpretation. Here, we describe a lentiviral barcode-based multiplexing approach, CellTag Indexing, which uses predefined genetic barcodes that are heritable, enabling cell populations to be tagged, pooled, and tracked over time in the same experimental replicate. We demonstrate the utility of CellTag Indexing by sequencing transcriptomes using a variety of cell types, including long-term tracking of cell engraftment and differentiation in vivo. Together, this presents CellTag Indexing as a broadly applicable genetic multiplexing tool that is complementary with existing single-cell technologies. Electronic supplementary material The online version of this article (10.1186/s13059-019-1699-y) contains supplementary material, which is available to authorized users.

Summary Oligosaccharide, a typical danger‐associated molecular pattern (DAMP), has been studied and applied as plant defence elicitor for several years. Here, we report a novel oligosaccharide, mannan oligosaccharide (MOS) with a degree of polymerization of 2–6, which was hydrolysed from locust bean gum by a newly reported enzyme, BpMan5. The MOS treatment can significantly enhance the generation of signalling molecules such as intracellular Ca 2+ and reactive oxygen species. Subsequent defence events like stomata closure and cell death were also caused by MOS, eventually leading to the prevention of pathogen invasion or expansion. Transcriptional expression assay indicated that MOS activated mitogen‐activated protein kinase cascades in tobacco and rice via different cascading pathways. The expression levels of the defence‐related genes PR‐1a and LOX were both up‐regulated after MOS treatment, suggesting that MOS may simultaneously activate salicylic acid and jasmonic acid‐dependent signalling pathways. Furthermore, liquid chromatography‐mass spectrometry analysis showed that MOS led to the accumulation of four phytoalexins (momilactone A, phytocassane A, phytocassane D, and phytocassane E) in rice seedling leaves within 12–24 h. Finally, MOS conferred resistance in rice and tobacco against Xanthomonas oryzae and Phytophthora nicotianae , respectively. Taken together, our results indicated that MOS, a novel DAMP, could trigger multiple defence responses to prime plant resistance and has a great potential as plant defence elicitor for the management of plant disease.

Single-cell technologies have seen rapid advancements in recent years, presenting new analytical challenges and opportunities. These high-throughput assays increasingly require special consideration in experimental design, sample multiplexing, batch effect removal, and data interpretation. Here, we describe a lentiviral barcode-based multiplexing approach, 'CellTag Indexing', where we transduce and label samples that can then be pooled together for downstream experimentation and analysis. By introducing predefined genetic barcodes that are transcribed and readily detected, we can reliably read out sample identity and transcriptional state via single-cell profiling. We validate and demonstrate the utility of CellTag Indexing by sequencing transcriptomes at single-cell resolution using a variety of cell types including mouse pre-B cells, primary mouse embryonic fibroblasts, and human HEK293T cells. A unique feature of CellTag Indexing is that the barcodes are heritable. This enables cell populations to be tagged, pooled and tracked over time within the same experimental replicate, then processed together to minimize unwanted biological and technical variation. We demonstrate this feature of CellTagging in long-term tracking of cell engraftment and differentiation, in vivo, in a mouse model of competitive transplant into the large intestine. Together, this presents CellTag Indexing as a broadly applicable genetic multiplexing tool that is complementary with existing single-cell technologies.

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Vesicular trafficking plays a crucial role in protein localization and movement, signal transduction, and multiple developmental processes in eukaryotic cells. Vesicle fusion is the final and key step in vesicle-mediated trafficking and mainly relies on SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors), the regulators including SM (Sec1/Munc18) family proteins, Rab GTPases and exocyst subunits. Verticillium dahliae is a widespread soil fungus that causes disruptive vascular diseases on a wide range of plants. To date, no genes involved in vesicular fusion process have been identified and characterized in V. dahliae. The recent publication of the draft genome sequence of V. dahliae allowed us to conduct a genome-wide identification, phylogeny and expression profile of genes encoding vesicular fusion components. Using compared genomics and phylogenetic methods, we identified 44 genes encoding vesicle fusion components in the V. dahliae genome. According to the structural features of their encoded proteins, the 44 V. dahliae genes were classified into 22 SNAREs (6 Qa-, 4 Qb-, 6 Qc-, 1 Qbc- and 5 R-types), 4 SM family proteins, 10 Rab GTPases and 8 exocyst proteins. Based on phylogeny and motif constitution analysis, orthologs of vesicle fusion component in filamentous fungi were generally clustered together into the same subclasses with well-supported bootstrap values. Analysis of the expression profiles of these genes indicated that many of them are significantly differentially expressed during vegetative growth and microsclerotia formation in V. dahliae. The analysis show that many components of vesicle fusion are well conserved in filamentous fungi and indicate that vesicle fusion plays a critical role in microsclerotia formation of smoke tree wilt fungus V. dahliae. The genome-wide identification and expression analysis of components involved in vesicle fusion should facilitate research in this gene family and give new insights toward elucidating their functions in growth, development and pathogenesis of V. dahliae.

In direct lineage reprogramming, transcription factor (TF) overexpression reconfigures Gene Regulatory Networks (GRNs) to convert cell identities between fully differentiated cell types. We previously developed CellOracle, a computational pipeline that integrates single-cell transcriptome and epigenome profiles to infer GRNs. CellOracle leverages these inferred GRNs to simulate gene expression changes in response to TF perturbation, enabling network re-configuration during reprogramming to be interrogated in silico. Here, we integrate CellOracle analysis with lineage tracing of fibroblast to induced endoderm progenitor (iEP) conversion, a prototypical direct lineage reprogramming paradigm. By linking early network state to reprogramming success or failure, we reveal distinct network configurations underlying different reprogramming outcomes. Using these network analyses and in silico simulation of TF perturbation, we identify new factors to coax cells into successfully converting cell identity, uncovering a central role for the AP-1 subunit Fos with the Hippo signaling effector, Yap1. Together, these results demonstrate the efficacy of CellOracle to infer and interpret cell-type-specific GRN configurations at high resolution, providing new mechanistic insights into the regulation and reprogramming of cell identity.

Complex gene regulatory mechanisms underlie differentiation and reprogramming. Contemporary single-cell lineage tracing (scLT) methods use expressed, heritable DNA barcodes to combine cell lineage readout with single-cell transcriptomics enabling high-resolution analysis of cell states while preserving lineage relationships. However, reliance on transcriptional profiling limits their adaptation to an ever-expanding tool kit of multiomic single-cell assays. With CellTag-multi, we present a novel approach for profiling lineage barcodes with single-cell chromatin accessibility without relying on co-assay of transcriptional state, paving the way for truly multiomic lineage tracing. We validate CellTag-multi in mouse hematopoiesis, characterizing transcriptional and epigenomic lineage priming across progenitor cell populations. In direct reprogramming of fibroblasts to endoderm progenitors, we use CellTag-multi to comprehensively link early cell state with reprogramming outcomes, identifying core regulatory programs underlying on-target and off-target reprogramming. Further, we reveal the Transcription Factor (TF) Zfp281 as a novel regulator of reprogramming outcome, biasing cells towards an off-target mesenchymal fate via its regulation of TGF-β signaling. Together, these results establish CellTag-multi as a novel lineage tracing method compatible with multiple single-cell modalities and demonstrate its utility in revealing fate-specifying gene regulatory changes across diverse paradigms of differentiation and reprogramming.