GSM1565995: DC_MTB (TAB-Seq); Homo sapiens; Bisulfite-Seq
DNA from infected and non-infected DCs was extracted using the PureGene DNA extraction kit (Gentra Systems). Genomic DNA was spiked with 0.25% of M. SsI methylated lambda DNA and 0.25% of 5hmC spike-in control II (where all cytosines were 5hmC) and then sonicated to 200-500bp with a Covaris ultrasonicator. Next,  b-glucosyltransferase (bGT) was used to introduce a glucose onto 5hmC, generating b-glucosyl-5-hydroxymethylcytosine (5gmC) to protect 5hmC from further TET oxidation. After blocking of 5hmC, all 5mC is converted to 5caC by oxidation with an excess of recombinant Tet1 protein. Bisulfite treatment of the resulting DNA then converts all C and 5caC (derived from 5mC) to uracil or 5caU, respectively, whereas the original 5hmC bases remain protected as 5gmC. Thus, following sequencing, only the 5hmC that were protected from bisulfite conversion will be read as cytosine bases. After the treatment, bisulfite conversion and library preparation were performed following a protocol identical to that for MethylC-Seq libraries.