CUT&Tag analysis uncovered Pou4f1-bound DNA elements in E16.5 retinas

To understand how Pou4f1 functions in RGC lineage specification and subtype formation, we performed “Cleavage Under Targets & Tagmentation” (CUT&Tag) analysis using a rabbit anti-Pou4f1 antibody and embryonic 16.5 (E16.5) retinal cells to generate barcoded PCR libraries that are enriched for Pou4f1-mediated binding. In parallel, rabbit IgG was used as a negative control for peak calling analysis, and rabbit anti-H3K9ac antibody was used to mark active enhancers and promoters. Overall design: Four retinas isolated from wildtype mouse embryos at E16.5 were pooled, and then dissociated using papain dissociation system (#LK003150, Worthington Biochemical Corporation, Lakewood, NJ). The CUT&Tag library was prepared using CUT&TAG-IT Assay Kit (#53160, Active Motif, Carlsbad, CA) following manufacturer's protocols. The libraries were pooled evenly and subjected to the paired-end 75-cycle sequencing on an Illumina NextSeq 550 System using High Output Kit v2.5 (#20024907, Illumina, San Diego, CA).