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Neural multiomics Q&A
Increased single-cell sensitivity is a constant goal in the next-generation sequencing field.
In our on-demand webinar, "Scaled, high-fidelity electrophysiological, morphological, and transcriptomic cell characterization," Dr. Brian Lee discussed how scientists in his lab are improving transcriptomic data using Takara Bio's highest sensitivity SMART-Seq technology in their neuronal Patch-seq workflow.
Continue reading for insights from the Q&A session or register to watch the on-demand webinar.
Can you add any other measurements to Patch-seq, like whole genome amplification or any type of epigenetic information?
Dr. Lee: Epigenetic information is possible, since we do have the nucleus. We've recently talked about this at our Institute, but the effort has not yet started. However, we do acknowledge that there are epigenetic mechanisms there that we can use to further classify cell types.
What is the percentage overlap between the clusters and the met space?
Dr. Lee: I don't know the exact percentage of what the overlap is, but we are able to take the framework from our RNA-seq platform and map our cells onto that. What we have found is that when the nucleus is included, that we do get a nice alignment with particular types, subclasses and transcriptomic types, to the RNA seek platform.
How many cells does it require information for before you consider to have enough information and can start making scientific conclusions?
Dr. Lee: I think it depends on what type of neuron you're looking at, the diversity of those neurons, and then what modality that you really want to try to incorporate. For example, inter neuron types within the visual cortex, we have six or seven different subclasses and as you saw, 10 15, 20 different met types.
So really to make those, I think that you need hundreds, if not thousands of cells. On the flip side, if we were to go to a subcortical region and we start to see some initial results of the diversity, and the diversity between those types is not as great, then maybe we can make that work with a hundred cells. I think that's something that you have to start evaluating as you collect the data.
Any tips for maintaining a clean ePhys rig for Patch-seq experiments?
Dr. Lee: That's one thing we really had to spend a bit of time to do. We invited the folks from molecular biology to look over our rigs to see how suitable they were for obtaining molecular biology information, and they were actually astonished to see how dirty our rigs are compared to a molecular biology wet lab.
They gave us some tips and techniques, and we just tried to be as clean as possible. We wear gloves, we wipe down all surfaces. Anything that we touch, we change gloves multiple times a day. We use ethanol to make sure our hands are clean. We have brought hoods into the ePhys lab. So we have hoods just adjacent to the ePhys rigs. As we collect the data, we're able to quickly bring it to the hood and deposit it and reduce any type of air contamination or RNase contamination.
View single-cell RNA-seq data from libraries generated with our SMART-Seq Single Cell PLUS Kit, a complete kit for cDNA synthesis and library preparation.
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