An optimized strategy for cloning-based locus-specific bisulfite sequencing PCR

In this methods paper, we describe a successful strategy to investigate locus-specific methylation by cloning-based bisulfite sequencing. We cover sample handling, DNA isolation, DNA quality control before bisulfite conversion, bisulfite conversion, DNA quality control after bisulfite conversion, in silico identification of CpG islands, methylation-independent bisulfite sequencing PCR (BSP) assay design, methylation-independent BSP, cloning strategy, sequencing and data analysis. Methods that are described nicely elsewhere will not be covered in detail. Instead, the focus will be on tips/tricks and new methods/strategies used in this protocol, including quality control assessment of the DNA before and after bisulfite conversion and a pooled cloning strategy to reduce time, costs and effort during this step. In addition we comment on dealing with bias and improving overall protocol efficiency.