Scientists at KOTAI Biotechnologies share how they guarantee nuclei and cell high quality for dependable single-cell sequencing outcomes
Single-cell sequencing allows researchers to achieve detailed, in-depth details about cell sorts, gene expression and gene regulation inside a organic system. The approach has been used throughout a spread of analysis areas, together with immunology, oncology, an infection, and neuroscience, to offer unprecedented perception into organic mechanisms of well being and illness, biomarker discovery and therapeutic improvement.
Utilizing the newest sequencing applied sciences, researchers can achieve substantial knowledge from their samples. It’s important to make sure high-quality nuclei preparation to maximise the success of those experiments.
On this SelectScience interview, we converse with KOTAI Biotechnologies CEO, Dr. Kazuo Yamashita; scientist, Dr. Masakazu Ishikawa; and basic supervisor, Hiroyuki Shimizu. We be taught in regards to the firm’s cutting-edge 10x Genomics® single-cell sequencing workflow, how the DeNovix CellDrop™ FL Cell Counter allows them to make sure pattern high quality for single-nucleus transposase-accessible chromatin (ATAC) and RNA sequencing, and the way this work is offering deeper perception into the immune system.
Scientists at KOTAI Biotechnologies use single cell genomics and AI to research a spread of analysis areas together with immunology, oncology, an infection, and neuroscience
Profiling immune response: Deeper perception with single-cell genomics
KOTAI Biotechnologies specializes within the mixture of synthetic intelligence (AI) and immunology analysis for biomarker and drug discovery. Collaborating with researchers from academia and the pharmaceutical trade, the corporate implements state-of-the-art know-how to offer a broader understanding of the immune system. Present initiatives are investigating customized most cancers immunotherapies, autoimmune illness and COVID-19.
The power to profile immune responses on the single-cell stage can present deep characterization and vital insights into the underlying mechanisms of illness, in addition to the influence of therapeutics. This strategy allows researchers to establish particular cell sorts, states or regulatory mechanisms which may be liable for these illness states. “We now have to seek out which cells are certainly in control of the pathogenicity, for instance. To that finish, we have to see cell by cell,” says Yamashita. “For this reason single-cell sequencing is a really essential strategy for our analysis.”
Making certain pattern high quality for single-cell sequencing
KOTAI Biotechnologies is an authorized supplier of 10x Genomics single-cell sequencing workflows – an modern know-how adopted by researchers world wide that permits the processing of tens of 1000’s of single cells in a single run.
Earlier than samples are processed, high quality should be checked. This step is often carried out utilizing cell viability assays to make sure solely residing cells are processed. “First, we have to see the situation of the cells and see if the pattern preparation labored or not. After which if it really works, we should always assess if the variety of residing cells is sweet sufficient for the single-cell sequencing,” Yamashita explains. “If the pattern has so many lifeless cells, then we should always take away these first in order that we are able to use 100% of the ten,000 slots for the residing cells.”
KOTAI Biotechnologies use the DeNovix CellDrop™ FL Automated Cell Counter of their single cell sequencing workflow
A single nucleus strategy
When working with samples the place it’s tough to isolate particular person cells, equivalent to frozen mind tissue, a single nucleus sequencing strategy could also be used. For instance, single nucleus RNA-seq (snRNA-seq) and transposase-accessible chromatin-seq (ATAC-seq) allow the respective evaluation of transcriptomes and chromatin accessibility, cell by cell. Collectively, these strategies present data on regulatory mechanisms that management gene expression.
Library preparation for each strategies requires the isolation of nuclei from cell or tissue samples. This isn’t at all times a straightforward process – samples can include a whole lot of particles and intact cells should even be eliminated. To make sure pattern high quality, KOTAI Biotechnologies carry out acridine orange and propidium iodide (AO/PI) twin fluorescence counting utilizing the DeNovix CellDrop™ FL Automated Cell Counter. This methodology gives clear advantages over guide cell counting, and a recent study by the group identifies AO/PI as extra correct than trypan blue. “We now have confirmed that the AO/PI is extra correct, by assessing it when it comes to the viability and likewise by utilizing the construction of the cells,” says Yamashita.
Confirming cell viability and cell depend is especially vital as a result of samples acquired by the staff at KOTAI Biotechnologies might not have all been ready the identical method – samples are ready by totally different researchers.
A key characteristic that attracted the staff at KOTAI Biotechnologies to buy the CellDrop™ FL is its skill to totally automate AO/PI cell counting course of – one thing which isn’t potential on all cell counters. “They’re extra correct and are totally automated, so are very handy and there aren’t any experimenter’s biases. So, we principally use the CellDrop for the cell counting,” says Yamashita.
Picture of cells taken utilizing the DeNovix CellDrop™ FL Automated Cell Counter. Washed PBMC used for example to depend the variety of cells by AO/PI. Greens and reds indicated the dwell and lifeless cells, respectively. Picture courtesy of KOTAI Biotechnologies
A much bigger image of immunology
Along with high-throughput genomic knowledge from single-cell sequencing, complementary research like protein-protein interactions are serving to the staff at KOTAI Biotechnologies to achieve an even bigger image of immunology. “We’re simply part of that massive image, however very essential, as a result of the cell is the essential unit. And since we’re an AI and immunology firm, we’re additionally making an attempt to develop a brand new single-cell know-how. However on the identical time, we’re additionally accumulating the several types of knowledge to mix them collectively to see the large image of immunology.”
Trying to the long run, Yamashita highlights the significance of having the ability to achieve extra data from single cells and the position the know-how will play on this. “The development of the know-how is to get extra and totally different varieties of knowledge from the only cell. So not simply gene expressions, not simply protein expressions, the floor proteins or the intracellular proteins, and likewise the epigenetic data from the cells,” Yamashita explains. “For this reason the standard of the cell could be very essential.”
Efficient pattern preparation will stay basic to success on this work. “The pattern preparation is essential and the measurement of the situation of the cells will likely be fairly essential for the downstream evaluation and downstream experiments,” Yamashita concludes.
Wish to be taught extra in regards to the single nuclei evaluation protocol? Download the technical note, for free, here >>
