Mapping genome-wide binding profile of ChCUC1 by DNA Affinity Purification sequencing (DAP-seq)

The mechanism underlying CUC genes regulating leaf marginal patterning remains largely unknown. To this end, we employed DAP-seq to identify the downstream regulating genes of ChCUC1. The aim of this project is to map genome-wide CUC genes binding sites. Overall design: A genomic DNA library is first prepared according to general library construction methods that include fragmentation of genomic DNA followed by ligation with Illumina-based sequencing adaptors. Oxford genomic DNA were used for library construction. To allow for high-throughput expression of affinity-tagged ChCUC1, ChCUC1 open reading frames (ORFs) are transferred to the Gateway-compatible pIX-HALO expression vector, which contains an N-terminal HaloTag affinity tag sequence. The pIX-HALO-CUC construct is then expressed using plant (wheat germ)-based in vitro transcription/translation coupled system. Purified proteins are then combined with the adaptor-ligated genomic DNA library and can bind to genomic DNA fragments in a sequence-specific manner. After washing away the unbound fragments, the beads are boiled to denature the protein and release the DNA into solution. This DNA is then PCR-amplified to attach multiplexing index sequences and enrich for TF-bound fragments. Samples are pooled and the resulting final library is sequenced using an Illumina sequencing platform.