Amplicon of TFAM CpG island of Homo sapiens: Open Heart Surgery Atrial Appendage
DNA was extracted from patient tissue as described above. DNA was bisulfite-treated using the EZ DNA Methylation-Lightning Kit (Zymo Research), per manufacturers instructions. Briefly, sulfonation of DNA results in the conversion of unmethylated cytosines into uracil, which are subsequently converted to thymine. Primers were designed for the CpG island of TFAM; primer set 1 amplified the 3 end and primer set 2 amplified the 5 end of the CpG island (Supplemental Table 2). Bisulfite DNA was prepared for sequencing using a 2-Step PCR amplification process (Bernstein et al, 2015). Step-1 PCR adapters included a base pairing region (~23 bp) and an overhang Illumina adapter arm (~33 bp). Bisulfite DNA from all 50 patients was PCR amplified using Step-1 primers utilizing Platinum Taq DNA Polymerase (Thermo Fisher), run on 2% agarose gels, extracted through a QIAquick Gel Extraction Kit (Qiagen), and DNA purified. DNA was then further amplified using Step-2 Illumina barcoded adapters and prepared using a 300-cycle MiSeq Reagent Micro Kit v2 (Illumina, San Diego, CA). PCR amplicons were sequenced on the MiSeq with paired-end (PE) 250 base pair reads. Sequencing was performed through the West Virginia University Genomics Core.