Specific pathogen free chicken eggs

Specific-pathogen-free chicken eggs (Charles River Laboratories, catalog 10100329) were incubated in a humidified incubator at 38 °C and collected at embryonic day 4 or 5 (Hamburger Hamilton stage 24 and 26). Eyes were enucleated and washed in cold PBS, and a sheet of RPE from the posterior eye chamber was collected directly into cold ATAC-seq lysis buffer (Active Motif cat. 53150). The RPE from two embryos were collected per biological sample. Biological replicates were carried out in duplicate and ~100,000 nuclei were loaded per reaction. Library preparation was carried out per manufacturer’s instructions (Active Motif cat. 53150), and final libraries were validated on the Agilent Bioanalyzer before sequencing on a lane of HiSeq 4000. Each sample was sequenced to a minimum depth of 80 million 150 base pair paired end reads. Raw reads were quality trimmed using trim galore with the parameters –clip_R1 16 –clip_R2 18 –three_prime_clip_R1 6 –three_prime_clip_R2 4^{57 (link),58} and high-quality reads were aligned to the chicken genome (GRCg6a) using Bowtie 2^{59 (link)} with the parameters –very-sensitive -k 5 -p 40^{59 (link)}. Biological replicates were collapsed and visualized using Integrative Genomics Viewer^{46 (link)}.