Methylation Analysis of miR-5088-5p Promoter

After genomic DNA was extracted according to a standard phenol-chloroform extraction method, bisulfite modification of genomic DNA was performed using an EZ DNA methylation kit (Zymo Research, USA). The methylation analysis of the miR-5088-5p promoters was performed using MSP primer pairs covering the putative transcriptional start site in the 5′ CpG islands with 1 μℓ of bisulfite-treated DNA as template and JumpStart Red Taq DNA Polymerase (Sigma-Aldrich Company, MO, USA) for amplification as previously described [27 (link)]. DKO (DNMT1(−/−), DNMT3B (−/−) double knockout in HCT116 cells) as unmethylation control, IVD (in vitro methylated DNA) as methylation control, and ddH2O as PCR negative control were used. Primers of the miR-5088-5p promoter region across the upstream from −225 to 34 from mature form sites (Supplementary Fig. S3A). The sequences of the unmethylation and methylation miR-5088-5p promoter primers used for MSP and qMSP are listed in Table 2. MSP amplification was performed on bisulfite treated samples and normalized using the Alu element. Real-time PCR was performed by a CFX96TM real-time system (Bio-Rad, Hercules, CA, USA). Alu primer sequence information is previously described [28 (link)].