Zebrafish Photoreceptor Transcriptome Allogeneic Analysis And Validation

Background and Purpose:With the development and improvement of second-generation and third-generation sequencing technologies,high-throughput sequencing technologies such as genome sequencing,transcriptome sequencing and single-cell sequencing are becoming less and less expensive and more and more popular.How to extract effective information from mass data is becoming more and more challenging.Traditional bioinformatics is basically the use of different computational methods to analyze and process data.Therefore,we hope to with analyzing the transcriptome of five photoreceptors in zebrafish retina,in the process of exploring the differential genes of zebrafish photoreceptor cells,establish a high-throughput sequencing data processing method.And combine with the classical biotechnologies such as CRISPR-Cas9 knockout and immunohistochemistry to verify and support the conclusions of traditional bioinformatics.Methods:Transcription,as an important part of the genetic central dogma,is the basis of regulating cell metabolism,protein synthesis and proliferation and the transcriptome contain this important information.In this study,we use various open sourced transcriptome sequencing analysis packages in R language,such as DESeq2 and Limma,to analyze the biological functions and signal channels of these specific genes of photoreceptors.Then we combined the references and the enrichment analysis to screen out the photoreceptors' differential genes which worthy of our attention.Finally,the basic biological verification of these differential genes is carried out,such as CRISPR-Cas9 knockout,immunohistochemistry,phenotypic analysis and so on.To explore the differences in genes that really affect photoreceptors and to verify the accuracy of transcriptome calculations.Results:The experimental results showed that only a small part of the genes in zebrafish retina were differentially expressed,and most of them were located on the photosensitive membrane discs and related to light signal conversion.The knockout of the screened differentially expressed genes by CRISPR-Cas9 showed that most of the differentially expressed gene deletions did not cause significant phenotypic changes.But immunohistochemistry experiments using the retina showed that the loss of some of these genes caused changes in the proportion of photoreceptors.Among them,RORCa loss will cause a decrease in the proportion of UV cones,while Nr2f1 a may be beneficial to the formation of red cones.Through our preliminary work,we screened and verified certain genes related to the development and maintenance of zebrafish retinal photoreceptors.We hope that this work will help with gene therapy and stem cell therapies,as well as high-throughput sequencing data analysis.