Hairpin Whole Genome Bisulfite Sequencing Analysis Of Hemimethylation Profiles In Cells

DNA methylation is a common epigenetic modification that plays a key role in a variety of biological processes,such as X chromosome inactivation,gene expression,embryonic development,genomic imprinting,and cancer development.Embryonic stem cells are a class of pluripotent stem cells with unlimited self-renewal capacity and differentiation potential.It has been shown that DNA methylation is closely related to the pluripotency and differentiation ability of embryonic stem cells.Bisulfite sequencing is the gold standard for DNA methylation detection.However,during heavy bisulfite conversion,DNA double strands open and separate,and data analysis makes it difficult to clarify the methylation status of both strands of the same DNA source.In order to analyze the symmetry of DNA methylation between the two strands in cells,this study established the Hairpin whole genome bisulfite sequencing(HPWGBS)system with reference to the existing literature,using hairpin adapter to link the positive and negative strands of DNA together.The methylation status of the two strands of DNA originating from the same DNA duplex was sequenced.In this study,the efficiency of bisulfite transformation was firstly examined,and the results showed that the highest transformation efficiency was achieved at a bisulfite concentration of 0.5× and a BS transformation time of 9 h.In this study,the ratio of hairpin junction and sequencing junction was optimized to achieve a high level of connection efficiency of hairpin junction.The amplified libraries were purified by magnetic beads and separated by agarose gel electrophoresis,so that the ligation efficiency of hairpin junctions in the obtained products reached more than 90%.To study the status of DNA hemimethylation in embryonic stem cells,DNA obtained by ultrasonic interruption and enzymatic interruption were subjected to HPWGBS library building and sequencing to obtain 100 G of data,respectively.The analysis revealed that the ligation efficiency of hairpin junction was 91% and the genome matching efficiency was more than 78%.the overall coverage of Cp G sites was 95%,of which >70% of Cp G was measured more than 5 times,indicating that the system was relatively complete in detecting Cp G sites in the genome.The analysis of DNA methylation levels of the double strands showed that the global methylation level in ESCs was 26.30%,the positive strand methylation level was 7.82% and the negative strand methylation level was 7.74%.The differentiation process of ESCs is accompanied by the adjustment of methylation status of pluripotency genes and genes related to germ layer differentiation.To clarify the patterns of changes in methylation status of specific gene regulatory regions(mainly Cp G islands)during differentiation of ESCs from the single cell level,studies were performed based on pluripotency genes(e.g.Fut4,Prmt5,Vgll4)and triterminal marker genes(e.g.Tubb3,Meis1,Pdgfra,Bmp4,Hand1,Gata6,Klf5,Sox17,Foxa2,Gsc,Cxcr4)of Cp G island sequences were designed as primers for heavy sulfite-PCR.Samples were collected from ESCs during mimicry differentiation and primordial-state-initial state(NPT)differentiation,genomic DNA was extracted,transformed with heavy sulfite and then used for BS-PCR.PCR products from each sample were mixed equimolarly and then enzymatically broken into fragments of about300 bp,and sequencing junctions were ligated for library construction to simultaneously detect DNA in multiple target regions in the same sample.methylation status of multiple target regions in the same sample.At present,this study can realize multiple pairs of primers put into the reaction system for BS PCR at the same time.In summary,this study 1)established and optimized the HPWGBS system and used it to study the distribution of DNA hemimethylation in ESCs,and 2)resolved the transition patterns of pluripotency and methylation of Cp G islands of differentiationrelated genes during random differentiation and NPT in ESCs.Thus,it provides a technical means to study the distribution and function of DNA hemimethylation in a variety of cells and tissues,and provides a reference to further understand the regulatory mechanisms during pluripotency exit or transition of ESCs.