Bisulfite genomic sequencing to uncover variability in DNA methylation: Optimized protocol applied to human T cell differentiation genes

Abstract

DNA methylation, which most commonly occurs at the C5 position of cytosines within CpG dinucleotides, is one of several epigenetic mechanisms that cells use to control gene expression. The importance of DNA methylation in a variety of biological processes (i.e., embryonic development, cellular proliferation and differentiation, chromosome stability) has led to a demand for a precise and efficient method to determine the exact DNA methylation status. Bisulfite genomic sequencing is regarded as a gold-standard technology for detection of DNA methylation as it provides a qualitative, quantitative and efficient approach to identify 5-methylcytosine at single base-pair resolution. To optimize the final results of the bisulfite genomic sequencing protocol, numerous modifications have been explored and have significantly improved the sensitivity and accuracy of this procedure. The aim of this methodological report is to give an overview of the bisulfite genomic sequencing protocol, discussing the critical methodological aspects. Since we are interested in studying the methylation status of specific genes involved in T cell development, we applied the bisulfite genomic sequencing to the study of the CD8A T cell co-receptor gene to determine whether the CGIs of this gene were subjected to methylation in different types of tissues. The results show that CD8A gene is differentially methylated depending on the tissue. In conclusion, we described a bisulfite genomic sequencing protocol that can be successfully used for the quantitative analysis of CpG island methylation of specific genes.