BD Rhapsody™ Single-Cell Analysis System Workflow: From Sample to Multimodal Single-Cell Sequencing Data

Ulbrich, Jannes; Lopéz-Salmerón, Vadir; Gerrard, Ian

doi:10.1007/978-1-0716-2756-3_2

Cited by 14 publications

(6 citation statements)

References 15 publications

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“…Sequencing was performed on an Illumina Novaseq 6000. Alignment to mm10/GRCm38 was performed using the BD Rhapsody Whole Transcriptome Analysis (WTA) pipeline 26 .…”

Section: Methodsmentioning

confidence: 99%

Bacterial ADP-heptose initiates a revival stem cell program in the intestinal epithelium

Goyal,

Guo,

Ranger

et al. 2024

Preprint

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The intestinal epithelium has an exceptional capacity to repair following injury, and recent evidence has suggested that YAP-dependent signaling was crucial for the expansion of Clu+ revival stem cells (revSCs) with fetal-like characteristics, which are essential for epithelial regeneration. However, neither the mechanism underlying where these revSCs emerge from nor the nature of the physiological cues that induce this revSC program, are clearly identified. Here, we first demonstrate that Alpk1 and Tifa, which encode the proteins essential for the detection of the bacterial metabolite ADP-heptose (ADP-Hep), were expressed by the stem cell pool in the intestinal epithelium. Treatment of intestinal organoids with ADP-Hep not only induced acute NF-κB pro-inflammatory signaling but also TNF-dependent apoptosis within the crypt, causing blunted proliferation and acute disruption of the crypt architecture, while also triggering induction of a revSC program. To identify the molecular underpinnings of this process, we performed single-cell RNA-seq analysis of ADP-Hep-treated organoids as well as lineage-tracing experiments. Our data reveal that ADP-Hep induced the specific ablation of the homeostatic intestinal stem cell (ISC) pool. Removal of ADP-Hep resulted in the rapid recovery of ISCs through dedifferentiation of Paneth cells, which transiently acquired revSC features and expressed nuclear YAP. Moreover, lineage tracing from Lyz1+ Paneth cells showed that ADP-Hep triggered Paneth cell de-differentiation towards pluripotent and proliferative cells in organoids. In vivo, revSC emergence in response to irradiation-induced injury was severely blunted in Tifa-deficient mice, suggesting that efficient epithelial regeneration in this model required detection of microbiota-derived ADP-Hep by the ALPK1-TIFA pathway. Together, our work reveals that Paneth cells can serve as the cell of origin for revSC induction in the physiological context of microbial stimulation, and that the transient loss of Alpk1-expressing ISCs is the initiating event for this regenerative process.

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“…Sequencing was performed on an Illumina Novaseq 6000. Alignment to mm10/GRCm38 was performed using the BD Rhapsody Whole Transcriptome Analysis (WTA) pipeline 26 .…”

Section: Methodsmentioning

confidence: 99%

Bacterial ADP-heptose initiates a revival stem cell program in the intestinal epithelium

Goyal,

Guo,

Ranger

et al. 2024

Preprint

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“…For all targeted transcriptomics and CITE‐Seq experiments, we used the microwell‐based single‐cell capture platform, BD Rhapsody™ system [8]. To evaluate the compatibility of cells stained with dye‐oligo conjugates for CITE‐Seq using the BD Rhapsody™ system, PBMCs were divided into two samples and stained with a 13‐plex ADT panel (Table 1) and a unique sample tag using a sample multiplexing kit (BD Biosciences) for 30 min at room temperature.…”

Section: Methodsmentioning

confidence: 99%

Flow cytometry analysis of protein expression using antibody‐derived tags followed by CITE‐Seq

Shi,

Fan,

Mehrpouyan

et al. 2023

Cytometry Pt A

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Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE‐Seq) is a single‐cell phenotyping method that uses antibody‐derived tags (ADTs) to quantitatively detect cell surface protein expression and generate transcriptomic data at the single‐cell level. Despite the increased popularity of this technique to study cellular heterogeneity and dynamics, detailed methods on how to choose ADT markers and ensuring reagent performance in biological relevant systems prior to sequencing is not available. Here we describe a novel and easy‐to‐use multiplex flow proxy assay in which multiple protein markers can be measured simultaneously using a combination of ADT reagents and dye‐oligo conjugates by flow cytometry. Using dye‐oligo conjugates with sequences complementary to the ADT reagents, we can achieve specific binding and evaluate protein marker expression in a multiplex way. This quality control assay is useful for guiding ADT marker choice and confirming protein expression prior to sequencing. Importantly, the labeled cells can be directly isolated based on the specific fluorescence from dye‐oligo conjugates using a flow cytometry cell sorter and processed for downstream single‐cell multiomics. Using this streamlined workflow, we sorted natural killer cells and T cells efficiently using only ADT and dye‐oligo reagents, avoiding the possibility of decreased marker resolution from co‐staining cells with ADT and fluorescent antibodies. This novel workflow provides a viable option for improving ADT marker choice and cell sorting efficiency, allowing subsequent CITE‐Seq.

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“…The BD Rhapsody platform is another commercial system used for scRNA-seq; however, unlike the 10× Genomics system, it utilizes magnetic beads rather than GEMs in a microfluidic device for cell isolation and barcoding [97][98][99]. Another notable characteristic of the BD Rhapsody system is that it is a microwell-based technology, whereas the 10× Genomics utilizes a droplet-based approach [99].…”

Section: Single-cell Rna Sequencingmentioning

confidence: 99%

Advancements in technology for characterizing the tumor immune microenvironment

Yan,

Ju,

Huang

et al. 2024

Int. J. Biol. Sci.

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Immunotherapy plays a key role in cancer treatment, however, responses are limited to a small number of patients. The biological basis for the success of immunotherapy is the complex interaction between tumor cells and tumor immune microenvironment (TIME). Historically, research on tumor immune constitution was limited to the analysis of one or two markers, more novel technologies are needed to interpret the complex interactions between tumor cells and TIME. In recent years, major advances have already been made in depicting TIME at a considerably elevated degree of throughput, dimensionality and resolution, allowing dozens of markers to be labeled simultaneously, and analyzing the heterogeneity of tumour-immune infiltrates in detail at the single cell level, depicting the spatial landscape of the entire microenvironment, as well as applying artificial intelligence (AI) to interpret a large amount of complex data from TIME. In this review, we summarized emerging technologies that have made contributions to the field of TIME, and provided prospects for future research.

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BD Rhapsody™ Single-Cell Analysis System Workflow: From Sample to Multimodal Single-Cell Sequencing Data

Cited by 14 publications

References 15 publications

Bacterial ADP-heptose initiates a revival stem cell program in the intestinal epithelium

Bacterial ADP-heptose initiates a revival stem cell program in the intestinal epithelium

Flow cytometry analysis of protein expression using antibody‐derived tags followed by CITE‐Seq

Advancements in technology for characterizing the tumor immune microenvironment

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