GSM2689857: Empty Vector_3_pool; Homo sapiens; OTHER
Genomic DNA was extracted using Puregene Gentra DNA extraction protocol. Genomic DNA extracted from tissue samples was spiked with methylation standards and sequentially digested with two restriction endonucleases recognizing CCCGGG sites in DNA. SmaI cleaved only unmethylated sites resulting in blunt ended fragments starting with 5’-GGG. Next, XmaI cleaved the remaining methylated sites creating 5’-CmeCGG overhang sequences. Exonuclease-deficient Klenow fragment DNA polymerase was then used to fill in the 3’ recesses left by XmaI and add 3’-dA overhangs to the ends of all fragments. Thus, specific signatures 5’-GGG and 5’-CCGGG were created at unmethylated and methylated CpG sites, respectively. Illumina sequencing adapters were then ligated to the enzyme-treated DNA; sequencing libraries were generated according to Illumina protocols and run on an Illumina HiSeq 2500 instrument at Fox Chase Cancer Center Genomics facility. Sequencing data were aligned to the rat genome (rn4), and the reads with unmethylated and methylated signatures were counted at individual CCCGGG sites. Methylation levels were calculated as the ratio of reads with the methylated signature to all reads mapping to each respective site. DNA methylation values at individual CpG sites were adjusted based on spiked in standards and filtered for the minimum sequencing depth of 100 reads.