Endogenous OCT4 interactome in hESC

In two widely-cited OCT4-protein interactome papers, Ding et al. (Ding et al., 2012) used murine ESC nuclear extract to identify OCT4 interactome in nucleus and Pardo et al. (Pardo et al., 2010) used whole cell lysate to identify OCT4 interactome in murine ESCs. In both papers, Benzonase, a genetically engineered endonuclease was employed to fragment genome DNA which significantly improved the enrichment efficiency of nuclear TFs. Since our focus was on cytosolic pool of OCT4, we used mild conditions for cell lysis and modified the Pardo’s method by omitting the addition of any nucleases to the lysis buffer. Such conditions not only retained the protein/RNA interactions, but also enabled precipitation of the nuclear TFs with genome DNA, and hence a relative enrichment of the cytosolic proteins and other soluble nuclear proteins (such as splicing factors) in the supernatant, and therefore allowed us to minimize the interference of the TF roles of OCT4 and to focus more on its novel RBP roles.