Single-Cell RNA Sequencing:

[Virtual Presenter] Good afternoon everyone, Single-Cell RNA Sequencing is a powerful tool and it has the potential to revolutionize research and provide us with a better understanding of the intricate relations between different biological molecules. Today, we will be discussing its techniques, discoveries, and potential. Let's get started..

[Audio] We will be exploring single-cell RNA sequencing. We will examine the techniques used in scRNA-seq, going over some recent findings from this technology, and considering its potential usage both presently and in the future. Our objective is to offer a complete overview of scRNA-seq and its positive effects for your research..

[Audio] ScRNA-seq is a powerful and fast-working method for examining gene expression within individual cells. It is widely used in various fields, such as genomics, developmental biology, immunology, and neurobiology. This presentation looks at current scRNA-seq techniques, discusses findings, and explores the potential applications. It also examines four papers that show how scRNA-seq can be used to study developmental biology and immunology. Finally, it looks at what future possibilities this technology holds for research and discovery..

[Audio] RNA-sequencing is a powerful tool to gain insight into gene expression at the cellular level. Going a step further, single-cell RNA-sequencing allows us to analyze differences between individual cells in a sample, opening up more possibilities for further understanding of cellular heterogeneity, including that of pancreatic cancer. With single-cell sequencing, researchers can discover unique features of individual cells in a sample, leading to further progress in understanding the various types of pancreatic cancer..

1. What is RNA-sequencing?. Faculty of Science. 5.

1. What is RNA-sequencing?. Faculty of Science. 6.

[Audio] Moving from bulk analysis of cells to single-cell analysis reveals a much more detailed and nuanced view of cells. This approach enables us to observe individual cellular phenotypes, which can be valuable for diagnostics and therapeutic applications. Additionally, it may uncover new cell types and tissues, and provide a greater insight into disease and drug effects..

[Audio] Single-cell RNA sequencing, or scRNA-seq, was first developed in the early 2000s and since then has seen rapid advances. It has become a popular method in diagnostic, biomedical, and drug discovery applications. Single-cell RNA sequencing has opened up a new way to examine gene expression on a cellular level. A large number of discoveries have been made possible by its use. It has aided in unlocking the secrets of gene expression and provided insights into various diseases, such as cancer. The possibilities of further discoveries and insights through single-cell RNA sequencing remain to be seen..

Nature paper: "mRNA-Seq whole-transcriptome analysis of a single cell" by Tang et al. Next-gen sequencing technology is powerful for ultra-high troughput transcriptome analysis BUT microgram amounts of RNA are needed 100 000's of cells Practically impossible to get under many important conditions (embryo research) Need for single cell resolution.

[Audio] Researchers have made impressive advances in technology since 2009, allowing them to accomplish single-cell RNA sequencing. They modified a single-cell whole-transcriptome amplification method, allowing them to amplify cDNAs over 3kb, with no bias. As a result, they developed a mRNA-Seq assay with a single mouse blastomere, making it possible to study the development of one mammalian cell..

[Audio] Single-cell RNA sequencing has seen great advancements in technology since 2009. Despite appearing simple, the RT step to convert mRNA to cDNA requires significant time and PCR primers must be adapted by adding an amine at the 5' end to prevent joining of double-stranded cDNA. Additionally, the SOLiD workflow should be considered. Through such modifications, single-cell RNA sequencing has become a valuable method for exploring gene expression in individual cells..

[Audio] Single-Cell RNA Sequencing (scRNA-seq) is a powerful technology that allows scientists to measure the expression of hundreds of thousands of genes in a single cell, as well as detect new splice isoforms and analyze the splice complexity of individual cells. In 2009, it was first used to validate its sensitivity in comparison to microarray studies, with 95% of genes having at least 5 reads and confirmed accuracy via real-time PCR. Ultimately, the technique was successful in detecting an impressive 71% of genes..

[Audio] The potential of single-cell RNA sequencing continues to be promising. Through this technology, scientists have been able to measure copy number accuracies with minimal decrement and simultaneously analyze expressed isoform. Furthermore, there were also notable discoveries of previously unknown splice junctions. While there are still a few limitations, this technique is a huge breakthrough for the field of genomics..

[Audio] Single-cell RNA sequencing has advanced considerably in the last twenty years, providing more precise and accurate measurements of gene expression. This technique has a range of uses, from organoid models and stem cell development to intercellular interactions and spatial transcriptomics. In addition, it has allowed for the categorization of cell types, discovery of new genes, and discovery of cell-specificity of disease-causing genes in tumor tissues. The versatility of scRNA-seq has lent itself to a better understanding of the molecular basis of life..

[Audio] Single-cell RNA sequencing has become an essential tool in molecular biology for analyzing gene expression. Protocols allow researchers to isolate and collect thousands of cells from a tissue sample, and sequence the transcriptome of each cell. This has drastically changed our comprehension of cellular heterogeneity, pinpointing rare cell types that would not have been found with traditional methods. It has also provided further understanding of diseases and gene expression..

[Audio] A diagram of a single-cell sequencing process is presented here. This technique permits sequencing of transcripts in individual cells, allowing for an unparalleled level of insight into the characteristics and activities of cells. It has allowed us to differentiate between cell types and comprehend how they interact with each other in a molecular fashion. Studies using single-cell RNA sequencing have already resulted in major advances in a variety of areas, like diseases and pharmaceuticals..

Faculty of Science. 17. Svensson et al Nature Protocols 2018.

[Audio] Plate-based SMART-seq is a method developed for single-cell RNA sequencing. It provides an economical and effortless solution for researchers from various disciplines and expertise. It can be performed with minimal technical requirements and used in rush projects. Moreover, it is suitable for long-term storage and freezing, and can be coupled with robots to allow for high throughput analysis. Additionally, this method can fit in with automation systems for large-scale studies. Another advantage is that it allows for control of the global environment around each cell, which makes it a great tool for researchers to investigate single-cell transcriptomes..

Drop-seq. Faculty of Science. 19. Svensson et al Nature Protocols 2018.

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[Audio] Microfluidic chips have been developed in recent years, thanks to advances in technology. These chips are useful for separating cells in a solution and measuring them through single-cell RNA sequencing. This method has allowed researchers to explore the genetic makeup of cells and explore their potential applications in medical therapies or bioengineering. The chip is able to sort, count and measure individual cells to identify their genetic information, as illustrated in the diagram..

[Audio] Drop-seq is an advanced technique for single-cell RNA sequencing, utilizing microfluidics to form tiny droplets. Generally, each droplet is made up of either a cell or a bead, with very few of them containing both. This allows for a precise sequencing process, uncovering valuable insights into the complexities of the single-cell transcriptome..

[Audio] Advancements in the area of single-cell gene expression analysis has created a range of new possibilities to discover unknown cellular states. Single-cell RNA sequencing is being used commonly and it is offered in different commercial formats. This text explores three of the most extensively used methods; Microwell-based SeqWell, Celsee Genesis, and the recently-introduced SeqWell Celsee solution. Every method is efficient, but each has its own characteristics and drawbacks, so it is important to take time and assess each system before choosing one..

Combinatorial indexing. Faculty of Science. 24. Cao et al Science 2017.

[Audio] Single-cell RNA sequencing is the process of looking at gene expression in individual cells. It is a powerful technique that has enabled researchers to gain deeper insights into cell biology and disease diagnosis than ever before. By analyzing the expression of a single-cell’s genes, researchers can gain a much better understanding of how cells react to different environmental stressors or drug therapies. The table highlighted here represents the potential to sequence at increasingly larger scales, from an individual cell up to larger clusters of cells, using this powerful technology. In combination with other analytical tools, single-cell RNA sequencing can open doors to understanding both healthy and diseased states of cells." Single-cell RNA sequencing is a groundbreaking technology that is revolutionizing our understanding of cell biology and disease. It allows researchers to analyze the expression of individual cells' genes, giving us unprecedented insights into how cells respond to different environmental conditions and treatments. This table is a visualization of the potential of single-cell sequencing; it shows us the ability of this technology to analyze samples at increasingly large scales, from a single cell to a cluster of cells. This data demonstrates how single-cell RNA sequencing, combined with other analytical techniques, can provide a detailed view of both healthy and diseased states of cells..

[Audio] Single-cell RNA sequencing offers a powerful new tool to accurately differentiate between different cell types within the same sample. By gathering data on the expression of thousands of genes, it is possible to compare cell types even when they are highly similar. This will help researchers identify new cell types and gain a better understanding of the underlying biological processes..

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[Audio] Single-Cell RNA Sequencing is an effective technique for examining the molecular composition of a single cell. It allows for much more detailed data than traditional methods which average out data from multiple cells. This article will look at three improvements on the basic single-cell RNA sequencing procedure: snRNA-seq, Spatial transcriptome analysis and EasySci full-length transcription scRNA-seq. As well, the latest version of single-cell RNA sequencing, Quartz-seq2, will be discussed. Through these advancements, many interesting findings have been made, as well as potential uses for single-cell RNA sequencing..

[Audio] Single-cell RNA sequencing has revolutionized our capacity to detect and quantify the genes expressed in single cells. It has made significant discoveries and is expected to provide more insights into our genetic characteristics and the progression of diseases. This presentation will discuss the various methods, potentials, and potential applications of this technology..

[Audio] Single-cell analysis is a powerful way to examine the complexity of organs and tissues. New research has used this technique to explore brain tissue in elderly people. Examining samples from individuals younger and older than 60, researchers noticed a substantial number of T cells infiltrating the old neurogenic niches, something that was nonexistent in younger individuals. This implies that these newly identified T cells may be related to aging and could be involved in neurodegeneration..

[Audio] Single-cell RNA sequencing has provided new insight into the graying of tissue functions with age. By analyzing cells in the subventricular zone of young and aged mice, it has become possible to understand how these alterations can affect tissue performance. These approaches also enable us to gain a further understanding of how adjustments in cell composition can modify communication between cells..

scRNA of the entire SVZ. Faculty of Science. 33. Cell-cell interactions.

[Audio] Single-cell RNA sequencing has completely changed the way biology is studied. Instead of looking at whole tissues, researchers can now study individual cells, deepening our understanding of the inner workings of organisms. This technology has revealed a wealth of new details about gene expression, disease, and the potential for personalized medicine. Through microfluidic chip technology, researchers can now load single cells with RT reagents and form emulsion GEMs - with up to 10 thousand cells in a single run. This is an extraordinary development that will have far-reaching applications in medical and biological research..

[Audio] After the lysis of the cells, the gel beads are dissolved to release barcoded RT oligonucleotides, which are then used to reverse-transcribe the polyadenylated mRNA. The cDNAs produced all contain the same barcode, allowing the sequencing reads to be mapped back to their original source. This process is at the core of single-cell RNA sequencing..

[Audio] Analysis of the neurogenic niche revealed eleven distinct cell types. Remarkably, the T-cell population was entirely comprised of cells from old mice and was especially prominent in the old subventricular zone. This suggests a possible role for the T-cell population in aged brains..

[Audio] Our research has revealed the intricate relationship between aging and the numbers of NSCs, NPCs and neuroblasts in the brain's neurogenic niche, along with dynamic interaction between T cells and NSCs. This indicates that both T cells and NSCs interact to influence each other in older brains. By understanding this relationship, we can gain insight into how to better care for elderly populations in the future..

[Audio] Using the Smart-seq v4 technique, researchers are examining the characteristics of T cells in aging brains. This technique offers a comprehensive insight into the immune system's activity in older brains, enabling researchers to better comprehend the influences of age. Moreover, it can be used to identify potential new treatments to improve elderly people's quality of life..

[Audio] Single-cell sequencing technology has revealed intriguing findings regarding the activity of immune cells in aged organisms. In the case of T cells, cells collected from elderly sources showed increased expression of the cytokine IFNγ, a phenomenon not noted in blood samples. Additionally, research has demonstrated that various cell types in the neurogenic niche are strongly susceptible to IFNγ. These studies clearly indicate the potential of single-cell sequencing to uncover novel biological knowledge..

[Audio] Studies have shown that with aging, T cells are more active within the neurogenic niche. This activity has been linked to a decline in the niche's capacity to generate new neural stem cells, or NSCs. Further research has revealed that IFNγ, a molecule which T cells produce, is the cause of the decrease in NSCs. Thus, it has been concluded that T cells affect the health of the neurogenic niche as we age, and this must be taken into account when developing potential treatments..

[Audio] Sziraki et al. have uncovered a new set of genes expressed in human pericyte cells through their study. Utilising single-cell RNA sequencing, the research team was successful in identifying markers for distinguishing pericyte cells. This finding gives researchers a powerful way to investigate the interaction between endothelial and mesenchymal cells in human tissues..

[Audio] Single-cell RNA Sequencing has been very important in discovering new genes linked to vasculature and neurodegenerative diseases such as Alzheimer's. It has been revealed that pericytes - cells located in the walls of blood vessels in the brain - have critical roles in the development of these diseases. As research progresses and more is uncovered, single-cell RNA sequencing is affording scientists greater amounts of data to grasp possible factors that lead to the growth of vascular and neurodegenerative diseases..

[Audio] Single-cell RNA sequencing technologies enable to examine the human brain at a molecular level. It provides a unique approach to analyze the basis of physiology and functionality. With this technology, it is possible to sequence individual cells which can unlock the molecular aspects of the distinct cell types that make up the human brain. This could open new doors to comprehending neurological diseases, complications, and treatments..

[Audio] Single-Cell RNA Sequencing is an incredibly powerful technique for studying gene expression in cells. It enables us to accurately measure the levels of thousands of genes across different cell types, and with new techniques, even rarer cell types can be detected. This data can then be used to systematically characterise cell-type-specific markers. With this data, we can gain new insights into how cells behave and interact with each other, helping to advance our understanding of biology..

[Audio] EasySci is a Reverse Transcription approach that is both high throughput and low cost. It utilizes indexed oligo-dT primers and random hexamer primers to achieve cell-type specificity, allowing for full gene body coverage with unprecedented insight into a single cell's gene expression..

[Audio] Cell type-specific markers have become critically important for the accurate analysis of brain tissue. Similar to immunohistochemical (IHC) markers that are employed to identify distinct types of neurons, these markers can be utilized to detect the placement of cells as well as how they are affected by different pathological alterations..

[Audio] Using single-cell RNA sequencing, we have been able to gain a more in-depth understanding of the cellular makeup of the human brain. We recently conducted an analysis of brain samples from six individuals with Alzheimer's disease and six control individuals. We used known markers to compare the pericyte genes to the other cell types in order to identify distinct vascular cell types. Through utilizing expression levels as a metric, we found that genes with expression greater than 700 transcripts per million were markers for the pericytes, while genes with expression less than 400 transcripts per million identified the second cell type..

[Audio] We have identified a set of five marker genes through single cell RNA sequencing which enable us to accurately and specifically identify pericytes in the human brain. Of these five marker genes, two are highly specific to pericytes while the other three cross over several other cell types in the brain. This discovery is of great significance for research into brain physiology and pathology as pericytes have wide implications for many different cell types. It will now allow us to gain further insight into the connections between different cells of the brain and any associated diseases..

[Audio] The Fly Cell Atlas provides a comprehensive single-nucleus transcriptomic atlas of the adult fruit fly, a potentially groundbreaking approach to studying single-cell transcriptomes. Developed by Li et al in 2022, it was used to map the transcriptional changes involved in ageing, with a view to gaining valuable insights into the molecular changes associated with the ageing process..

[Audio] Single-cell RNA sequencing has drastically changed our understanding of gene regulation in model organisms such as Drosophila. Research on these small genomes has revealed essential mechanisms and pathways, with 75% of genes that cause human disease being discovered in the fly. Advances in single-cell genomic analyses of cells and tissues have recently improved our understanding of cell-typing clustering pipelines and models, thanks to the decreased cost and improved performance of droplet-based and plate-based techniques. Crowd-sourced gene annotations have enabled us to accurately annotate different cell types. This concludes our presentation on single-cell RNA sequencing - its techniques, findings, and potential..