Investigation of DNA methylation at Chr7 alpha satellite DNA

To determine the methylation levels of alpha satellite DNA on Chr 7 57999639-58000359, genomic DNA was extracted from HEK293, HCT116 and HCT116 DNMT1 hyphomorphic cells using the QIAamp DNA Mini Kit (Qiagen), and bisulfite converted using EZ DNA Methylation-Lightning Kit (Zymo Research) following manufacturer's instructions. The bisulfite treated DNA was then used for PCR amplification using HotStarTaq DNA Polymerase (Qiagen) using primers with cell-type specific barcodes in the first 6 nucleotides:Cell line, Fw primer (5’ – 3´), Rv primer (5’ – 3´)HEK293, CAG ATC GTT ATT GGA TAT TTG GAG TAT TTT GAG GTT TAT GG, CAG TAC AAA CCA TCC TAA CTA ACA AAA TAA AAC CCHCT 116, ACA TGT GTT ATT GGA TAT TTG GAG TAT TTT GAG GTT TAT GG, ACT AGT AAA CCA TCC TAA CTA ACA AAA TAA AAC CCHCT116 DNMT1-/-, CTT GTA GTT ATT GGA TAT TTG GAG TAT TTT GAG GTT TAT GG, CTG TTA AAA CCA TCC TAA CTA ACA AAA TAA AA CCCPCR products were resolved on an 8% acrylamide gel, followed by extraction and clean-up using the NucleoSpin Gel and PCR Clean-up (Macherey-Nagel). The products were mixed at an equimolar ratio and paired-end sequening was conducted on an Illumina HiSeq2000 system (Novogene Bioinformatics Technology Co., Ltd., Beijing, China, www.novogene.cn).