GSM7061150: control pUC19, lambda phage, and T4-phage, no_TET_no_deam; Cloning vector pUC19; Escherichia phage T4; Escherichia phage Lambda; OTHER
In vitro derived controls for all other samples. 1 ng of sheared DNA input (consisting of fully unmodified C pUC19 DNA, fully 5mCpG modified Lambda control gDNA, and fully 5hmC-modified T4 phage gDNA) was used for each condition. DNA was added to a 20 μL reaction containing 1x NEB CutSmart buffer (50 mM Potassium Acetate 20 mM Tris-acetate, 10 mM Magnesium Acetate, and 100 μg/ml BSA), 0.04 nM UDP-glucose, and 10 U T4-βGT. The glucosylated DNA was then purified with SPRI magnetic beads (Beckman-Coulter) using a 1.2x left-sided selection. The purified DNA was then in a 50 μL reaction containing 1x NEB EM-Seq TET buffer (50 mM Tris pH 8.0, 1 mM DTT, 5 mM sodium-L-ascorbate, 20 mM αKG, 2 mM ATP, and 50 mM ammonium iron (II) sulfate hexahydrate) and 16 μg TET2 (NEB). The reaction was then incubated at 37°C for 80 min. Following oxidation, 0.8 U of Proteinase K (NEB) was added and the mixture was incubated for 30 min at 37°C. The oxidized DNA was then purified with SPRI magnetic beads (Beckman-Coulter) using a 1.2x left-sided selection. The no TET control was carried through mock oxidation reactions without TET enzyme. The DNA was then purified with SPRI magnetic beads (Beckman-Coulter) using a 1.2x left-sided selection. The purified oxidized sample was then end-prepped and ligated with the same protocol as above except for the adapters used, which was different for each method (no deamination, TAPS, TAPS-β: C adapters, BS-Seq: 5mC adapters, DM-Seq: 5pyC adapters). BS deamination, A3A deamination, pyridine borane deamination, or a no deamination control were then performed.