By Derek Vargas, Scientific Affairs, MedGenome Inc
Single cell sequencing is a cutting-edge technique used in molecular biology that enables the sequencing of the transcriptome of individual cells. In traditional bulk sequencing techniques, RNA is extracted from a large group of cells, and then sequenced as a whole. However, single cell sequencing allows researchers to analyze the genetic material of individual cells, providing a much more detailed and precise understanding of the diversity and heterogeneity of cell populations.
Single cell sequencing involves several steps, including isolating individual cells from a sample, lysing the cells to release their genetic material, amplifying the RNA to generate enough material for sequencing, and then sequencing the material using high-throughput sequencing technologies. There are several different types of single cell sequencing, each with its own strengths and limitations.
Single cell sequencing has many potential applications in basic research and clinical settings. It can be used to study complex biological processes such as embryonic development, cancer progression, and immune system function. It can also be used in research settings to identify rare cell types or genetic mutations that may be missed using traditional sequencing methods. MedGenome offers end-to-end single cell sequencing services using the 10X Genomics platform. Our extensive experience with single cell work allows us to process many samples with fast turnaround time.
Single Cell Sequencing at MedGenome
Despite the many advantages of single cell sequencing, there are also several challenges associated with this technique. Some of these challenges include:
- 1. High cost: Single cell sequencing can be expensive, as it requires specialized equipment and reagents, as well as significant computational resources for data analysis.
- 2. Low throughput: Single cell sequencing is a time-consuming process that can only sequence a limited number of cells at a time, which can limit the statistical power of the analysis.
- 3. Difficulty processing fresh samples: Many research labs are not able to process tissue samples quickly while cells are still viable. This may impact overall data quality
- 4. Limited availability of fresh samples: Single cell sequencing generally requires high quality fresh/frozen samples. This is often not possible for scientists who focus their studies on retrospective research using biobanked samples.
As a leading provider of single cell sequencing services, MedGenome has developed solutions to many of these problems. Using a combination of skilled scientists and automation, we can support large scale projects while keeping costs low. We also have protocols in place to help researchers preserve their precious samples until they can ship them to our labs. This has allowed MedGenome to process hundreds of single cell samples every year.
10X Genomics Single Cell Flex Kit
As I mentioned, a major limitation of single cell sequencing is that it can be difficult for researchers to preserve samples for processing. There are also researchers doing retrospective studies and getting new samples is impossible. 10X Genomics has recently released a new Single Cell Flex kit which addresses these issues. With this new Flex kit, cells are fixed and permeabilized and can be safely stored or transported without compromising data quality. Once ready to proceed, samples are hybridized to probe sets and may be processed individually (singleplex workflow) or pooled with up to sixteen samples in a single lane of a Chromium chip (multiplex workflow). During GEM generation the probe sets are ligated and extended to incorporate unique barcodes. Libraries are then prepared, sequenced, and analyzed using 10x Genomics Cell Ranger and other bioinformatics tools.
The ability to fix cells and tissues will help scientists who aren’t able to immediately process their samples for single cell sequencing. It is even possible to stain the cells with Totalseq antibodies prior to fixation. The cells can then be stored for months at -80 oC. This will benefit researchers doing longitudinal studies where samples are expected to be collected over a long period of time. In this case the samples can be fixed, frozen, and then shipped once the entire experiment is completed.
The Single Cell Flex kit also allows profiling of FFPE tissues. This workflow uses the Miltenyi Gentlemacs Dissociator (available at MedGenome) to dissociate FFPE scrolls into single cell suspensions. Since the Single Cell Flex kit is probe-based, it allows for a pseudo-transcriptome profiling of these highly degraded samples and allows researchers to gain insights into the cell populations of tissues that may have been collected several years ago.
MedGenome has always been an early adopter of single cell sequencing technologies. We started offering single cell gene expression, and have expanded over the years to support multiomic analysis of samples- including CITE-seq, immune profiling, and ATAC-seq. We have also adopted technologies for tissue dissociation and nuclei isolation. Our goal is to make single cell sequencing technologies available and affordable to genomics research labs, and to support research projects regardless of sample limitations or experiment complexity. Our team is excited about the new Single Cell Flex kit and expect to be offering this service very soon.
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