Epignome kit

Genomic DNA was isolated from 5mg of plaque tissue, using the MasterPure™ DNA Purification Kit (Epicentre) according to the manufacturer’s instructions. Bisulfite treatment of genomic DNA was then performed using the Zymo EZ DNA Methylation Lightning Kit. The bisulfite-treated DNA was purified on a spin column and used to prepare the sequencing library using the EpiGnome™ Kit (Epicentre). Paired-end sequencing with 150-bp read lengths was performed on Illumina HiSeq X10 instrument. Methylation analysis was performed using Bismark (v0.17.0). In summary, FASTQ files were quality-filtered and adapter sequences trimmed using Trimmomatic. A bisulfite-converted UCSC hg19 reference genome file was generated using Bowtie, and the EpiGnome library sequence data were aligned to the reference genome. Methylation information was extracted for those DGEs identified from the RNA-seq analyses. The promoter region was defined as 1500bp before and 500bp after the TSS of each gene. Fisher test was used to identify statistic significant changes on the methylation ratios.

Genomic DNA was isolated from the control and the DAC-treated CT26 cells and PDX tumors using a QIAamp genomic DNA Kit (Qiagen, Stockach, Germany) according to the instructions. Bisulfite treatment of genomic DNA was performed using a DNA Methylation Lightning Kit (Zymo EZ, CA, USA). Briefly, 200–500 ng of purified genomic DNA was treated with the Zymo Lightning Conversion Reagent in a thermal cycler for 8 min at 98 °C, followed by 60 min at 54 °C. The bisulfite-treated DNA was purified on a spin column and used to prepare the sequencing library using an EpiGnome Kit (Epicentre, Wisconsin, USA). In this procedure, bisulfite-treated single-stranded DNA was random-primed using a polymerase able to read uracil nucleotides to synthesize DNA that contained a specific sequence tag. The 3′ ends of the newly synthesized DNA strands were then selectively tagged with a second specific sequence, which resulted in doubly tagged DNA molecules with known sequence tags at their 5′ and 3′ ends. The EpiGnome libraries were diluted and loaded onto the cBot DNA Cluster Generation System. After the cluster generation was completed, the flow cell was transferred to the HiSeq 3000 System for sequencing using 75 bp paired-end reads. Additional sequencing could be completed for higher coverage when necessary. The data were analyzed according to the GO database and KEGG database.