Single mouse/human embryonic stem cells, gametes and early embryos were picked into the lysis buffer (20 mMTris, 2 mM EDTA, 20 mMKCl, and 0.3% TritonX-100) using a mouth pipette. Genomic DNAs were released after treatment with proteinase (QIAGEN) at 50°C for 3 h. To label the 5fC adequately, 150 mM malononitrile (J&K) was added directly to the single cell lysate, which was allowed to react at 37°C for 20 h with continuously shaking in a thermomixer (Eppendorf, 850 r.p.m.). For the control group, same volume of H2O was used instead of malononitrile. To obtain the whole genome 5fC profiles from the single cells, MALBAC technique was applied to amplify the malononitrile-labeled genomic DNAs with minor modifications(Zong et al., 2012). Briefly, 11 rounds of quasi-linear pre-amplification and 15 cycles of exponential amplification were performed to obtain sufficient DNA for library construction. Sequencing libraries were constructed under the instruction of NEBNext Ultra DNA Library Prep Kit for Illumina (NEB), and the final libraries were amplified using the DeepVent DNA polymerase (3'-5' exo-, NEB).