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===Preparation of gDNA for TAB-seq (protocols taken directly from publication) === [[File:TAB-seq.jpg | 300px]] *'''(1) Glucosylation and Oxidation of Genomic DNA''': Glucosylation reaction was performed in a 50 μl solution with 50 mM HEPES buffer (pH 8.0), 25 mM MgCl2, 100 ng/μl ''sonicated genomic DNA with spike-in control'', 200 μM UDP-Glc, and 1 μM wild-type βGT. The reaction was incubated at 37°C for 1 hr. After the reaction, the DNA was purified by a QIAquick Nucleotide Removal Kit (QIAGEN). The oxidation reaction was performed in a 50 μl solution with 50 mM HEPES buffer (pH 8.0), 100 μM ammonium iron (II) sulfate, 1 mM α-ketoglutarate, 2 mM ascorbic acid, 2.5 mM DTT, 100 mM NaCl, 1.2 mM ATP, 10 ng/μl glucosylated DNA, and 3 μM recombinant mTet1. The reaction was incubated at 37°C for 1.5 hr. After proteinase K treatment, the DNA was purified with Micro Bio-Spin 30 Columns (Bio-Rad) and then by a QIAquick PCR Purification Kit (QIAGEN). *'''(2) Bisulfite conversion of treated gDNA or untreated gDNA''': 500 ng–1 μg untreated or βGT/mTet1-treated (the same procedure as mouse ES/H1 cell) mouse cerebellum sample was applied to EpiTect Bisulfite Kit (QIAGEN) following the supplier's instruction. *'''(3) Library Generation''': 500 ng–1 μg treated genomic DNA was end-repaired, adenylated, and ligated to methylated (5mC) adapters (Illumina TruSeq Genomic DNA adapters) according to standard Illumina protocols for genomic DNA library construction, maintaining the proper molar ratios of adaptor to insert. Adaptor ligated fragments with 200–600 bp inserts were gel purified by 2% agarose gel electrophoresis and sodium-bisulfite treated using the MethylCode kit (Invitrogen). Bisulfite-treated adaptor-ligated DNA was amplified by PCR with PfuTurbo Cx Hotstart DNA polymerase. The number of PCR cycles used was determined by quantification of bisulfite treated adaptor-ligated DNA by qPCR (KAPABiosystems library quant kit for Illumina libraries) such that the final library concentration obtained was approximately 20 nM. Final sequencing libraries were purified with AMPure XP beads or 2% agarose gel electrophoresis and quantified by qPCR (KAPABiosystems library quant kit for Illumina libraries). Up to three separate PCR reactions were performed per sample.
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