Overview of CETCh-seq experimental method. (A) A schematic of the CETCh-seq approach is displayed. Cells are transfected with plasmids containing the Cas9 nuclease, gRNAs, and epitope tag donor constructs, leading to the homologous integration of the Flag tag, P2A linker sequence, and neomycin resistance gene at the 3′ end of the transcription factor in place of the endogenous transcription factor stop codon. Flag-tagged transcription factor and neomycin resistance genes are cotranscribed. Subsequently, a tagged transcription factor and a neomycin resistance protein are generated due to the P2A linker sequence. Cells are selected and colony-forming units are expanded for ChIP-seq experimentation using a Flag antibody. (B) HepG2 DNA-binding protein read enrichment tracks on the UCSC Genome Browser are given. The names of transcription factors are given. WT denotes transcription factor antibody experiments, while Flag Rep 1 and Flag Rep 2 are technical replicates using Flag antibodies for CETCh-seq experiments.
