The Principle of Single Cell Sequencing Technology and Its Development in the Biology Field (III)

3.2 Application of sc RNA-seq in the field of ophthalmology

The retina has a certain neuron diversity, which can be used to study neuronal differentiation and nervous system diversity. The retina is composed of various types of cells, such as ganglion cells, microglia cells, photoreceptor cells, etc. Due to its complex composition structure, conventional gene sequencing can no longer accurately reflect the specificity between retinal cells, but sc RNA-seq can overcome this difficulty and accurately reflect the gene expression of individual retinal cells. Transfected bipolar cells and Müller glial cells with GSP, collected GFP-positive cells by FACS, and applied large-scale Drop-seq to classify about 25,000 mouse retinal bipolar cells to obtain 15 bipolar cell subtypes. The matched molecular expression and cell morphology verified the accuracy of this classification method, and at the same time the method identified a variety of new genetic markers. The identification of genetic markers of individual retinal cell subtypes is conducive to the study of targets related to specific visual functions, enabling people to gain a deeper understanding of the source of cell function and cell heterogeneity.

Degeneration of the retina, such as age-related macular degeneration (AMD), is the result of damage to photoreceptor cells, which is also the main reason for the deterioration of vision in people in developed countries. The study found that the expression of transcription factors related to apoptosis is higher in patients with dry AMD, the expression of transcription factors related to angiogenesis is higher in patients with wet AMD, and all types of AMD have a cell-mediated immune response. Therefore, AMD may be a single disease with a common immune response.


3.3 Application of sc RNA-seq in the field of immunology research

The heterogeneity of the immune system helps to effectively defend against many different pathogens. In recent years, sc RNA-seq has been applied to the research of immune system. The response of immune cells to antigenic substances is characterized by complexity and heterogeneity. Lipopolysaccharide can activate Toll-like receptors in dendritic cells to trigger changes in the transcriptome. A new Th2 cell subpopulation can be identified by using sc RNA-seq to reveal the wide heterogeneity within the T helper 2 (Th2) cell population and marked with Cyp11a1 that regulates the steroid synthesis pathway. The technique of RNA transcription at the 5´-end can be used to construct a cDNA library and sequencing analysis of mouse bone marrow dendritic cells (BMDCs). The results showed that at the single cell level, dendritic cells response is highly heterogeneous. In some dendritic cells, many known genes that regulate the inflammatory response are in a fully activated state, while in other cells, they are only activated at a low level or not activated. The maturation state of protruding cells is related to the randomness of gene regulatory networks.

Although the application of SCS in the research of the immune system is relatively limited, SCS has shown strong potential in the immune cell subsets, detecting gene expression variability, differential splicing, and gene regulatory networks.


3.4 Application of sc RNA-seq in the field of animal breeding research

In juvenile in vitro Embryo Transfer (JIVET), scRNA-seq was used to screen out key genes that affect the maturation of lamb oocytes, and combined with real-time quantitative PCR technology to quantitatively verify the genes, namely, to study the regulation mechanism of oocyte maturation at the molecular level to improve the latent oocyte embryonic potential. And this experiment successfully found that MOS, RPS6KA1, CPEB1, ANAPC13 and CDK1 5 genes are extremely important for the maturation of lamb oocytes. JIVET uses oocytes on the ovary of young females as the source of eggs to cultivate embryos in vitro. The efficiency provides abundant genetic resources for livestock breeding.


Some studies have found that by analyzing and comparing transcriptomes, the ligand-receptor interaction signaling pathway for neural activity in the hypothalamus-pituitary-ovarian gonadal axis regulation of ovulation and follicle development plays an important role in regulating and screening out the production The related genes of lamb number enrich and supplement the information of sheep genome.


The RNA-seq technology was used to perform transcriptomics sequencing and data comparative analysis of yak GV and MII oocytes, and 4 767 differentially expressed genes were screened out. After analyzing the differential genes through KEGG pathway interaction, it was found that metabolic pathways play a key role in regulating gene up- and down- pathways during the maturation of oocytes,. RNA-seq technology was used to perform transcriptome sequencing and comparative analysis on the ovaries in the estrus of yak and central plain yellow cattle, and found that the cell adhesion process is one of the important conditions for distinguishing the physiological activities of the ovary of yellow cattle and yak. Moreover, there is a close relationship between the complement and coagulation cascade pathways and the reproduction and ovarian development of yak, which may be related to the special natural environment of the yak.


Although single cell separation is difficult, many single cell separation techniques can be applied to the separation and extraction of different types of cells to the greatest extent. Obtain single cells in an ideal state; sc RNA-seq can obtain in-depth analysis of heterogeneity between different single cells, gene expression networks, etc. by obtaining the full transcriptome expression profile from the single cell level; High-throughput, high-efficiency and low-cost sequencing technology can directly sequence RNA and discover new transcripts. Direct sequencing of RNA makes the results more accurate. Based on this comprehensive advantage, the application field of sc RNA-seq has gradually expanded from the research of early embryonic development of mammals to the treatment of human diseases in immunology, ophthalmology, tumors, etc., and achieved good results.



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