Library construction and oxidative bisulfite sequencing

For each hippocampal, cortical, and retinal sample, 1 μg of gDNA was brought up to 50 μl volume with 1× low-EDTA TE buffer and sheared with a Covaris E220 sonicator (Covaris, Inc., Woburn, MA) to an average base pair size of 200 using the following settings: intensity of 5, duty cycle of 10%, 200 cycles per burst, 2 cycles of 60 s, at 7 °C. The size of sheared products was confirmed by capillary electrophoresis (DNA D1000, Agilent). gDNA fragments were cleaned by an Agencourt bead-based purification protocol, after which gDNA was quantified (Qubit, Thermofisher Scientific). Two aliquots of 200 ng gDNA fragments were prepared in a 12 μl volume to which 1 μl of spike-in control DNA (0.08 ng/ul) with known levels of specific mC, hmC, and fC at individual sites was added. End repair, ligation of methylated adaptors (#L2V11DR-BC 1–96 adaptor plate, NuGEN, Tecan Genomics, Inc., Redwood City, CA) and final repair were performed according to manufacturer’s instructions (Ovation Ultralow Methyl-Seq Library System (NuGEN). Of the two DNA aliquots per sample, one was oxidized and then bisulfite-converted and the other only bisulfite-converted with the True Methy oxBS module (NuGEN) with desulfonation and purification. Twenty-two microliters of libraries was eluted from the magnetic beads. qPCR was used to determine the number (N) of PCR cycles required for library amplification. Bisulfite-converted samples were amplified for 7 cycles while oxidative bisulfite-converted samples were amplified for 11 cycles [95 °C—2 min, N (95 °C—15 s, 60 °C–1 min, 72 °C–30 s)]. Amplified libraries were purified with Agencourt beads and eluted in low-EDTA TE buffer. TapeStation HD1000 was used to validate and quantify libraries. Amplified libraries were normalized to a concentration of 4 nM and pooled, denatured, and diluted to 12 pM for sequencing on MiSeq (Illumina, San Diego, CA) according to manufacturer’s guidelines with the exception of a custom sequencing primer (MetSeq Primer) that was spiked in with the Illumina Read 1 primer to a final concentration of 0.5 μM.