H3K27ac HiChIP Profiling of Retinal Nuclei

H3K27ac HiChIP libraries were prepared as previously reported with minor modifications.¹⁴ (link) Briefly, following isolation of nuclei from frozen retinas as described above, ∼8 million nuclei from each sample were washed with nuclei isolation buffer from the diploid chromatin conformation capture (Dip-C) protocol and fixed with 2% paraformaldehyde at room temperature for 10 minutes.⁵¹ (link) Fixed nuclei were then washed twice with cold 1% bovine serum albumin in phosphate-buffered saline before resuspension in 0.5% sodium dodecyl sulfate and resumption of the published HiChIP protocol. Digestion was performed using the MboI restriction enzyme, and sonication was conducted using a Covaris E220 with 5 duty cycles, peak incident power of 140, and 200 cycles per burst for 4 minutes. The ab4729 ChIP validated antibody from Abcam was used to target H3K27ac. HiChIP libraries were sequenced with paired-end 75-bp reads on either an Illumina HiSeq 400 or Illumina NextSeq 550.