Em101

DNA methylation was examined by bisulfite conversion of extracted genomic DNA with a bisulfite conversion kit (#DP215, TIANGEN, China). The IFN-γ promoter was amplified by PCR using a methylation-specific PCR kit (#EM101, TIANGEN, China). Primer sequences were designed using Methyl Primer Express v1.0 (Applied Biosystems, USA), and Sanger sequencing was performed on the PCR products. Five individual PCR products were sequenced for each sample. Primers we designed are as follows: IFN-γ-BSP forward: 5’- TTTAGTATTTTGGGAGGTTAAGG-3’; reverse: 5’-ATCACCCAAACTAAAATACAATAAC-3’.

Potential CpG islands in the promoter region were predicted byMethPrimer(http://www.urogene.org/cgibin/methprimer/methprimer.cgi), and three amplification primers were designed based on the predictions therein. HCN4-1 (forward) 5’-TTTGTTTATAGTTTTTTGGTTATAGTT-3’, (reverse) 5’- TCCCAAAA TTTTCCTAAACCAC-3’; HCN4-2 (forward) 5’-TGTTTATAGTTTTTTGGTTAT AGTT-3’, (reverse) 5’-ACTCCCAAAATTTTCCTAAACC-3’, HCN4-3 (forward) 5’- TTGTTTATAGTTTTTTGGTTATAGTT-3’, (reverse)5’-TCCCAAAATTTTCCTAAA CCAC-3’. The extracted cellular DNA was treated with bisulfate (DP215-02, TIANGEN). The recovered DNA was specifically amplified using the above primers according to the MSP kit (EM101, TIANGEN). The PCR products were gel extracted and the recovered product was ligated to the T vector, then transformed into DH5abacteria and positive colonies were taken for sequencing. At least 10 separate clones were selected for sequencing. The sequencing results were compared with the original sequences using the QUMA (Quantification tool for Methylation Analysis) website(quma.cdb.riken.jp/top/index.html) to generate methylation site maps.