GSM7061127: control pUC19, lambda phage, and T4-phage, 50_mCpG_BS; Cloning vector pUC19; Escherichia phage T4; Escherichia phage Lambda; Bisulfite-Seq
In vitro derived controls for all other samples. To compare 5 different sequencing workflows (no deamination, TAPS, TAPS-β, BS-Seq, DM-Seq), 1 ng of sheared DNA input (consisting of fully unmodified C pUC19 DNA, 5mCpG modified Lambda control gDNA, and fully 5hmC-modified T4 phage gDNA) was used for each condition. The 5mCpG modified lambda control gDNA was one of 4 possibilities: fully unmethylated, fully CpG-methylated, fully GpC-methylated, and ~50% CpG-methylated. The ~50% CpG methylated DNA was made by mixing equal amounts of unmethylated and fully CpG-methylated lambda DNA. DNA was end-prepped and ligated with the same protocol as above except for the adapters used, which was different for each method (no deamination, TAPS, and TAPS-β: C adapters, BS-Seq: 5mC adapters, DM-Seq: 5pyC adapters). BS-Seq was performed using manufacturer instructions (Diagenode). Purified BS-Seq libraries were amplified using indexing primers (IDT) and HiFi HotStart Uracil+ Ready Mix (KAPA Biosystems) before purification over SPRI magnetic beads (0.8X) and ultimate characterization using a BioAnalyzer (High Sensitivity Kit, Agilent) and quantified (Qubit). DM-Seq was performed with the optimized protocol. A methylated copy strand was created. 1 μM fully-methylated copy primer was annealed in a total volume of 10 μL in CutSmart Buffer and 1 mM final concentration (individually) of dATP/dGTP/dTTP (Promega) and dmCTP (NEB). 1 μl or 8 units Bst polymerase, large fragment (NEB) was added and incubated for 30 min at 65°C. The 5hmCs were then glucosylated with 40 μM UDP-Glucose and 1 μL or 10 units of T4 Phage β-glucosyltransferase (NEB) for 1 hour at 37°C in a final volume of 20 μL. Incompletely copied or uncopied fragments were degraded with 1 μL or 10 units Mung Bean Nuclease (NEB) for 30 min at 30°C. After SPRI magnetic bead purification (1.2x), libraries were mixed with 0.5 μM MBP-M.MpeI-N374K and 160 μM CxSAM in carboxymethylation buffer (50 mM NaCl, 10 mM Tris-HCl pH 7.9, 10 mM EDTA) and incubated overnight at 37°C followed by  denaturation for 5 min at 95°C. 1 μL or 0.8 units of Proteinase K (NEB) was subsequently added and incubated at 37°C for 15 min. The samples were purified using SPRI magnetic beads (1.2x) and eluted in 1 mM Tris-Cl, pH 8.0. DNA was then subjected to snap-cooling and A3A deamination in a final volume of 50 μL as previously described before SPRI magnetic beads purification (1.2x). DM-Seq libraries were amplified using indexing primers (IDT) and HiFi HotStart Uracil+ Ready Mix (KAPA Biosystems) before purification over SPRI magnetic beads (0.8X). Libraries were then characterized using a BioAnalyzer (High Sensitivity Kit, Agilent) and quantified (Qubit). TAPS and TAPS-β were performed as previously described except for the source of TET enzyme (NEB, EM-Seq Conversion Module). 1 ng of sheared DNA input (consisting of fully unmodified C pUC19 DNA, 5mCpG-modified lambda phage gDNA, and fully 5hmC-modified T4 phage gDNA) was ligated to C-containing Y-shaped adaptors using the same protocol as described above (IDT). For TAPS-β, the ligated 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. This glucosylation step was omitted for TAPS. The purified DNA was then incubated 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 and input into a second round of TET oxidation. The oxidized DNA was added to a 5 μL reaction containing 600 mM sodium acetate (pH 4.3) and 1 M pyridine borane (Alfa Aesar). The reaction was incubated at 37 °C and 850 r.p.m. in a ThermoMixer (Eppendorf) placed in a chemical fume hood for 16 hrs and purified by Zymo-IC column (Zymo Research) with Oligo Binding Buffer (Zymo Research). The TAPS-β libraries were amplified using indexing primers (NEB) and HiFi Hotstart Uracil+ Ready Mix (KAPA Biosystems) before purification with SPRI magnetic beads (Beckman-Coulter) using a 1.2x left-sided selection. Libraries were ultimately characterized using a BioAnalyzer (High Sensitivity Kit, Agilent) and quantified (Qubit).