Effect of Cell cycle regulator CDK1 on Cell Differentiation (RNA-IP CDK1 inh KD)

We addressed the role of cell cycle regulator CDK1 during differentiation by either inhibiting its activity with the specific inhibitor RO3306 (10µM) or by knocking its expression down using specific shRNAs, during mesendoderm differentiation. Human pluripotent stem cells (H9 cells) were synchronized in their cell cycle and then induced to differentiate into mesendoderm for 24h. shRNAs against CDK1 were induced 4 days before starting the differentiation. The CDK1 inhibitor was added to the medium 8h after the induction of differentiation. At the end of the experiment, the cells were harvested and processed for total RNA extraction. RNA-IP was carried out using RiboCluster Profiler RIP-Assay Kit (MBL) following the manufacture's recommendations. Overall design: Four different conditions were analyzed: Undifferentiated (UD), Mesendoderm (E24), Mesendoderm + CDK1 inhibityor (E24RO), Mesendoderm without induction of shRNA_CDK1 with tetracycline (E24woTET), and Mesendoderm plus induction of shRNA_CDK1 with tetracycline (E2TET). Two biological replicates were generated for each condition.