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Simultaneous single-cell mRNA and epitope mapping of human CD4+ T-cells using the BD Rhapsody™ and the BD™ AbSeq platform.
Dr Ricardo C. Ferreira, University of Oxford
Description of the single-cell Multi Omics workflow that includes sample preparation, single-cell isolation and molecular barcoding, library preparation, sequencing and data analysis.
Christina Chang, PhD
“We were impressed with the straightforward workflow of the whole procedure of BD Rhapsody™ system from sample preparation to data analysis. Furthermore, when handling very small tissues (we used single mouse lymph nodes in our case), the BD Rhapsody™ is able to generate a multitude of in-depth information without waste of precious ex vivo material. Even without any prior experience in single-cell sequencing, we didn’t stumble on a single step with our first run and generated a massive data set, which was even workable for someone with only basic bioinformatics knowledge. The pipeline BD provides for processing the sequencing raw data is clearly arranged and easy to pick up. Data analysis with SeqGeq provides an excellent starting point for people already familiar with FlowJo and is a joy to work with. The ability to combine a massive amount of surface markers with targeted gene analysis on a single cell level while retaining the option to multiplex for a clean comparison between samples of different origin is a very strong method, which we certainly did not regret to pick up for our lab.”
As Alison Devonshire, Science Leader, Nucleic Acid Metrology, noted, "This new technology will allow us to further develop our single cell analysis capabilities to support our activities within engineering biology and precision medicine, for example for stem cell-based therapies or understanding the heterogeneity of cancer cells.”
Taken from on www.lgcgroup.com
A targeted multi-omic analysis approach measures protein expression and low abundance transcripts on the single cell level
Florian Mair, Jami R. Erickson, Valentin Voillet, Yannick Simoni, Timothy Bi, Aaron J. Tyznik, Jody Martin, Raphael Gottardo, Evan W. Newell, Martin Prlic
High throughput single-cell RNA sequencing (sc-RNAseq) has become a frequently used tool to assess immune cell function and heterogeneity. Recently, the combined measurement of RNA and protein expression by sequencing was developed, which is commonly known as CITE-Seq. Acquisition of protein expression data along with transcriptome data resolves some of the limitations inherent to only assessing transcript, but also nearly doubles the sequencing read depth required per single cell. Furthermore, there is still a paucity of analysis tools to visualize combined transcript-protein datasets.
Simultaneous mRNA and protein quantification at the single-cell level delineates trajectories of CD4+ T-cell differentiation
Dominik Trzupek, Melanie Dunstan, Antony J. Cutler, Mercede Lee, Leila Godfrey, Dominik Aschenbrenner, Holm H. Uhlig, Linda S. Wicker, John A. Todd, Ricardo C. Ferreira
The transcriptomic and proteomic characterisation of CD4+ T cells at the single-cell level has been performed traditionally by two largely exclusive types of technologies: single cell RNA-sequencing (scRNA-seq) technologies and antibody-based cytometry. Here we demonstrate that the simultaneous targeted quantification of mRNA and protein expression in single-cells provides a high-resolution map of human primary CD4+ T cells and reveals precise trajectories of canonical T-cell lineage differentiation in blood and tissue. We report modest correlation between mRNA and protein in primary CD4+ T cells at the single-cell level, highlighting the importance of including quantitative protein expression data to characterise cell effector function. This approach provides a massively-parallel, cost-effective, solution to dissect the heterogeneity of immune cell populations and is ideally suited for the detailed immunophenotypic characterisation of rare and potentially pathogenic immune subsets. This transcriptomic and proteomic hybrid technology could have important clinical applications to re-define differentiation and activation of tissue-resident and blood human immune cells.
A novel antibody targeting ICOS increases intratumoural cytotoxic to regulatory T cell ratio and induces tumour regression
Richard C.A. Sainson, Anil K. Thotakura, Miha Kosmac, Gwenoline Borhis, Nahida Parveen, Rachael Kimber, Joana Carvalho, Simon Henderson, Kerstin Pryke, Tracey Okell, Siobhan O’Leary, Stuart Ball, Lauriane Gamand, Emma Taggart, Eleanor Pring, Hanif Ali, Hannah Craig, Vivian W. Y. Wong, Qi Liang, Robert J. Rowlands, Morgane Lecointre, Jamie Campbell, Ian Kirby, David Melvin, Volker Germaschewski, Elisabeth Oelmann, Sonia Quaratino, Matthew McCourt
The immunosuppressive tumour microenvironment constitutes a significant hurdle to the response to immune checkpoint inhibitors. Both soluble factors and specialised immune cells such as regulatory T cells (TReg) are key components of active intratumoural immunosuppression. Previous studies have shown that Inducible Co-Stimulatory receptor (ICOS) is highly expressed in the tumour microenvironment, especially on TReg, suggesting that it represents a relevant target for preferential depletion of these cells. Here, we used immune profiling of samples from tumour bearing mice and cancer patients to characterise the expression of ICOS in different tissues and solid tumours. By immunizing an Icos knockout transgenic mouse line expressing antibodies with human variable domains, we selected a fully human IgG1 antibody called KY1044 that binds ICOS from different species. Using KY1044, we demonstrated that we can exploit the differential expression of ICOS on T cell subtypes to modify the tumour microenvironment and thereby improve the anti-tumour immune response. We showed that KY1044 induces sustained depletion of ICOShigh TReg cells in mouse tumours and depletion of ICOShigh T cells in the blood of non-human primates, but was also associated with secretion of pro-inflammatory cytokines from ICOSlow TEFF cells. Altogether, KY1044 improved the intratumoural TEFF:TReg ratio and increased activation of TEFF cells, resulting in monotherapy efficacy or in synergistic combinatorial efficacy when administered with the immune checkpoint blocker anti-PD-L1. In summary, our data demonstrate that targeting ICOS with KY1044 can favourably alter the intratumoural immune contexture, promoting an anti-tumour response.
Reliability of human cortical organoid generation.
Yoon SJ, Elahi LS, Pașca AM, Marton RM, Gordon A, Revah O, Miura Y, Walczak EM, Holdgate GM, Fan HC, Huguenard JR, Geschwind DH, Pașca SP.
The differentiation of pluripotent stem cells in three-dimensional cultures can recapitulate key aspects of brain development, but protocols are prone to variable results. Here we differentiated multiple human pluripotent stem cell lines for over 100 d using our previously developed approach to generate brain-region-specific organoids called cortical spheroids and, using several assays, found that spheroid generation was highly reliable and consistent. We anticipate the use of this approach for large-scale differentiation experiments and disease modeling.
Analyze the expression of hundreds of genes across tens of thousands of single cells in parallel
Whole Transcriptome Analysis Amplification Kit is now available !
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