Construction Of Transcriptome Maps Of Primary And Secondary Hair Follicles In Albas Cashmere Goats Using Single Cell RNA Sequencing Technology

Albas cashmere goat is one of the excellent varieties of cashmere goats that have been naturally and artificially bred,and the cashmere produced by it has extremely high economic value.There are primary and secondary hair follicles in the skin tissue of cashmere goats,and their growth and development form two different types of animal fibers: wool and cashmere.This is closely related to the significant differences in occurrence time,cell composition,and gene expression between the two types of hair follicles.Therefore,comparing the differences between primary and secondary hair follicles in cashmere goats and constructing two types of hair follicle growth and development regulation networks is the key to improving cashmere yield and quality.In this study,single cell transcriptome sequencing was used to analyze the primary and secondary hair follicles of cashmere goats.Through experiments,we successfully constructed transcriptome maps of primary and secondary hair follicles of Albas cashmere goats,compared the composition of primary and secondary hair follicle cell groups and gene expression differences,and also explored the temporal differentiation tracks of each hair follicle cell in the two hair follicle types.This has laid a solid foundation for the study of the regulatory mechanism for the growth and development of cashmere follicles.1.Isolation of hair follicle cells from Albas white cashmere goats.The preparation method of cashmere goat hair follicle single cell suspension was established.The cashmere goat hair follicle was digested in37 ℃ water bath for 30 minutes with trypsin 2.5 mg/ml,collagenase IV 1mg/ml,hyaluronidase 1 mg/ml and DNA enzyme 1 mg/ml mixed solution.2.Construction of the Hair Follicle Cell Composition Map of Albas White Cashmere Goat.The cells of cashmere goat hair follicles were analyzed by sc RNA seq technology,and 11692 high-quality single cell transcriptome data were obtained.Through t SNE clustering analysis,13 cell groups were obtained.The primary hair follicle is composed of 13 cell groups,while the secondary hair follicle is composed of 11 cell groups.Cluster 11 and Cluster 12 cell groups are unique to primary hair follicles.There is a significant difference in the proportion of various cell populations between primary and secondary hair follicles.Finally,screening marker genes were established for 13 hair follicle cell groups obtained through screening.3.Analysis of differentially expressed genes in primary and secondary hair follicles of Albas white cashmere goatsThe differential expression genes of various cell groups in secondary and primary hair follicles were compared and obtained.There are 1056,1242,1075,1182,953,1074,711,160,0,4,and 3 differentially expressed genes in the Cluster0,1,2,3,4,5,6,7,8,9,and 10 cell groups,respectively.GO functional enrichment analysis suggests that differentially expressed genes in various groups of hair follicle cells are mainly enriched in organelle,member bound organelle,and internal organelle entries on the Cellular Component.The differentially expressed genes are mainly enriched in RNA binding,heterocyclic compound binding,and organic cyclic compound binding entries on the Molecular Function;The differentially expressed genes are mainly enriched in the cellular protein complex disassembly entry on the Biological Process;Among them,the number of differentially expressed genes is the highest in the Organelle entry.KEGG functional enrichment analysis suggests that differentially expressed genes in various groups of hair follicle cells are mainly enriched in the Ribosome,Protein processing in endoplasic reticulum,Oxidative phonology,Carbon metabolism,and Parkinson disease pathways;Among them,the number of differentially expressed genes enriched in the Metabolic pathway is the highest.4.Construction of Single Cell Trajectories of Primary and Secondary Hair Follicles in Albas White Cashmere GoatsIn primary and secondary hair follicles,stromal cell 1 is located in the early stage of cell differentiation.As time went by,the cell group showed two different differentiation trends,one of which differentiated along the direction of hair follicle cortical cells;The other differentiates along the inner root sheath cell 1 direction of the hair follicle.In primary hair follicles,hair stromal cell 2 is located in the early stage of cell differentiation.As time passes,this cell group exhibits two different differentiation trends,one of which differentiates along the direction of hair follicle medulla cells;The other differentiates along the direction of the outer root sheath cells of the hair follicle.However,secondary hair follicle stromal cells 2 exhibit a chronomorphic differentiation relationship with outer root sheath cells,while there is no significant differentiation relationship with medullary cells.Comparing the differentiation of primary hair follicle stromal cells 2 in the directions of State 1 and State 3,GO enrichment analysis of differentially expressed genes showed that differentially expressed genes were mainly enriched in the system development,issue development,and multilateral organizational development entries on the Biological Process;On the vessel,member bound vessel,and extracellar region part entries on the Cellular Component.KEGG enrichment analysis showed that differentially expressed genes were mainly enriched in the Cysteine and methionine metabolism,Arginine and profile metabolism,Basal cell carcinoma,Hippo signaling pathway,and TGF-beta signaling pathway.