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.
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.
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.
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