GSM6858382: TI-0993-Diagnosis-AQ4039; Homo sapiens; OTHER
Around 1 ml of blood-derived plasma obtained at diagnosis was used to isolate cfcDNA with the QIAamp circulating nucleic acids kit (QIAGEN). NEBNext Enzymatic Methyl-seq Kit (New England Biolabs, NEB), following manufacturer's recommendations To prepare libraries for comprehensive methylome representation within a cfcDNA we used the NEBNext Enzymatic Methyl-seq Kit (New England Biolabs, NEB), following manufacturer's recommendations with minor modification that the cfcDNA was not sheared. The method utilizes 10 ng starting material of cfcDNA and relies on two step conversion of the cytosines. The first step uses TET2 oxidation of 5-methylcytosine that provides protection to the modified cytosines from conversion by APOBEC deamination, so only cytosines but not 5-methylcytosines are converted to uracil. Ultimately, cytosines are sequenced as thymines and 5-methylcytosines are sequenced as cytosines. The final 8 cycles of PCR library amplification were carried out using the NEB Unique dual index primer pairs (NEBNext multiplex oligos for Illumina).