Nextseq 500 500 high output kit v2

Phage-only samples were analyzed with 1 ng each of pooled methylated phage and T4-hmC gDNA (Fig. 2d). For all mammalian DNA samples, a total of 2 or 20 ng of sheared gDNA (~300 bp) was analyzed, containing the sheared methylated phage and T4-hmC spike-in controls (0.5% total by mass). In a total volume of 5 µL, 1–20 ng of the gDNA mixture was glucosylated using UDP-glucose and T4 β-glucosyltransferase (βGT, NEB) at 37 ºC for 1 hr. 1 µL of DMSO was added and the sample was denatured at 95 ºC for 5 min and snap cooled by transfer to a PCR tube rack pre-incubated at −80 ºC. Before thawing, reaction buffer was overlaid to a final concentration of 20 mM MES pH 6.0 + 0.1 % Tween, and A3A was added to a final concentration of 5 μM in a total volume of 10 µL. The deamination reactions were incubated under linear ramping temperature conditions from 4–50 ºC over 2 hrs. After deamination, the samples were prepared for Illumina sequencing using the Accel Methyl-NGS kit (Swift Biosciences). The resulting ACE-Seq libraries were sequenced at 1.9 pM with single-end mode on a NextSeq 500 sequencer (Illumina) using the NextSeq 500/500 High Output kit v2 (150 cycles). A more detailed step-by-step protocol for ACE-Seq, with added rationale and discussion, is provided as Supplementary Protocol 2.