New Strategy For Single Cell Epigenome Sequencing Based On Digital Microfluidics

With the rapid development of genome and transcriptome sequencing technology,more and more attention has been paid to the epigenome which connects the genome and its functional output.The epigenetic markers that have been discovered include DNA methylation,chromatin accessibility,histone modification,and so on.There are many methods for population cell epigenome sequencing,but bulk analysis may mask important but rare information and problems of heterogeneity between individual cells.Therefore,it is necessary to develop excellent single-cell epigenome sequencing methods in order to reveal deeper biological questions.Digital microfluidic is a newly developed discrete droplet manipulation platform in recent years.It has the advantages of large specific surface area,low reagent consumption,addressability,portability,etc.,and is expected to become a low-cost,low pollution,and automated single-cell epigenome sequencing platform.In this thesis,a digital microfluidic chip with hydrophilic spot was developed to capture single cells by the adhesion of hydrophilic spot to cells.Under the appropriate cell concentration and cell sedimentation time,the single-cell capture efficiency could reach 100%.And it had been experimentally verified that the method has no damage to cell viability,and has good droplet generation uniformity,excellent mixing efficiency,outstanding DNA recovery rate,high chip robustness,and controllable capture of a certain number of cells.On this basis,we used the chip for single-cell methylation sequencing,and developed a low-cost,high mapping rate,high coverage rate and high conversion rate single-cell methylome analysis method.Furthermore,using digital microfluidic chip without hydrophilic spot,combined with pooling and splitting technology,high-throughput single cell chromatin accessibility analysis was carried out.In conclusion,we developed an automated single-cell capture platform based on digital microfluidic chips and applied it to the sequencing analysis of single-cell epigenome,providing a new perspective for the study of epigenome.