Roche NimbleGen SeqCap Epi CpGiant (CpGiant)

One library (CpGiant_A_opt) was made using 1 µg of starting material according to the company’s specifications (SeqCap Epi CpGiant Enrichment kit, cat # 07138881001, Roche, Indianapolis, IN). DNA (1 µg) was sonicated using a Covaris S220 sonicator (Covaris, Woburn, MA) to obtain products of 180–220 bp. DNA was then end-repaired, A-tailed, ligated with methylated indexed-adapters to create a pre-capture DNA library and indexed using Illumina Index 6. The pre-capture library was bisulfite converted at 54 °C for 1 h using the Zymo EZ DNA Lightning kit (Cat # D5030, Zymo Research, Irvine CA). The bisulfite-treated pre-capture library was PCR-amplified for 11 cycles with HiFi HotStart Uracil + polymerase (Cat# KK2802, Kapa Biosystems, Wilmington, MA). 1 µg of the amplified, bisulfite converted library was then hybridized to the probe pool of fully, partially and unmethylated cytosines from both strands of DNA oligos at 47 °C for 72 h. Hybridized products were purified by capture with Capture Beads and PCR amplified for 16 cycles to create the final library. A second library (CpGiant_B_min) was made using 0.25 µg of starting material. DNA was then end-repaired, A-tailed, ligated with methylated indexed-adapters to create a pre-capture DNA library and indexed using Illumina Index 12. The library was then amplified for 13 cycles after post-bisulfite PCR amplification, 800 ng of library were used for hybridization and the post-capture PCR amplification cycles were kept at 16. Each library was clustered at 12 pM on a V3 paired-end read flow cell and sequenced for 100 cycles (PE100) on an Illumina HiSeq 2500.