Whole genome bisulfite-seq of CD4T-4
The extracted DNA was fragmented by S-series Focused-ultrasonicator (Covaris, Woburn, MA) using the 200-bp target peak size protocol. Fragmented DNA was then size selected (200600 bp) with an Agencourt AMPure XP bead-based (#A63880, Beckman Coulter, Brea, CA) size selection protocol. The DNA end repair step was performed with End-It DNA End-Repair Kit (#ER81050, Epicentre, Madison, WI). After the end-repair step, A-tailing (#M0202, New England Biolabs, Ipswich, MA) and ligation steps were performed to ligate the methylated adaptors. Bisulfite treatment of gDNA was performed using the MethylCode Bisulfite Conversion Kit (#MECOV50, ThermoFisher). Purified gDNA was treated with CT conversion reagent in a thermocycler for 10 min at 98 C, followed by 2.5 h at 640 C. Bisulfite-treated DNA fragments remain single-stranded as they are no longer complementary. Low-cycle (48) PCR amplification was performed with Kapa HiFi Uracil Hotstart polymerase enzyme (#KK2801, KAPA Biosystems, Wilmington, MA) which can tolerate uracil residues. The final library fragments contain thymines and cytosines in place of the original unmethylated cytosine and methylated cytosines, respectively.