DNA methylation protocol for analyzing cell-free DNA in the spent culture medium of human preimplantation embryos.

Cell-free DNA (cfDNA) in spent embryo culture media (SECM) provides prospects for noninvasive preimplantation genetic testing. Here, we present a post-bisulfite-adapter-tagging (PBAT)-based whole-genome DNA methylation sequencing protocol (SECM-PBAT) for human SECM cfDNA analysis. We describe steps for SECM lysis, bisulfite conversion and purification, preamplification by random priming, tagging adapter II, and library establishment. We then detail library quality control, sequencing, and bioinformatics analysis. This approach simultaneously detects chromosome aneuploidy and deduces the proportional contributions of cellular components. For complete details on the use and execution of this protocol, please refer to Chen et al. (2021).1.