Project description:To assess the effects of SOX9 depletion on active chromatin marks in hESC-derived cranial neural crest cells, we performed genome editing of the H9 hESC lines to tag SOX9 with FKBPV36, V5, and mNeonGreen. Upon differentiating edited hESC to cranial neural crest cells using an established protocol, addition of the degrader molecule dTAGV-1 results in SOX9 depletion. We then profiled the active chromatin mark H3K27ac by ChIP-Seq in undepleted CNCCs or CNCCs in which SOX9 was depleted for 3h or 24h. We also titrated SOX9 to four distinct levels with dTAGV-1 and assessed SOX9 binding by V5 ChIP-seq, as well as TWIST1 and TFAP2A binding by ChIP-seq.
Project description:To identify direct transcriptional targets of RFX6, we performed chromatin immunoprecipitation of HA epitope tagged RFX6 followed by massively parallel DNA sequencing (ChIP-seq). Using CRISPR/Cas9 gene editing, the HA epitope was inserted into the 3' end of the RFX6 gene in H9 hESC. Pluripotent cells were then differentiated into PDX1+RFX6+ pancreatic progenitors and endogenous RFX6-HA was immunoprecipitated with an anti-HA antibody. To eliminate background signal caused by non-specific antibody binding, a control experiment using wild-type H9 hESC was performed in parallel.
