Pyrogold reagents

A region of the mouse Apc gene around the Min mutation was amplified by polymerase chain reaction using the following primers 5’Bio-CCT CAA GGG GAA GTT TAG ACA GTT-3’ and 5’- GAT GGT AAG CAC TGA GGC CAA TA -3’. The 5’-biotinylated forward primer for the pyrosequencing reaction was isolated using streptavidin-coated Sepharose beads (Amersham Biosciences, Piscataway, NJ) and the PSQ 96 Sample Preparation kit (Biotage,Westborough, MA). The single-strand DNA template was incubated with sequencing primer 5’- CTG AGG CCA AT ACCT CG -3’. The sequencing by synthesis reaction of the complementary strand was performed on a PSQ 96MA instrument (Biotage) using PyroGold reagents (Biotage).

DNA isolation and telomere length measurements in the EpiPath were reported previously in Elwenspoek et al. (manuscript under review, Journal of Immunology). Methylation levels were measured at age-related CpGs in aspartoacylase (ASPA) (cg02228185), integrin alpha 2b (ITGA2B) (cg25809905), and PDE4CA (cg17861230) according to (22 (link)). In brief, unmethylated cytosine residues in each DNA sample were converted to uracil with a bisulfite treatment (EpiTect Bisulfite Kit, Qiagen, Venlo, Netherlands) and regions of interest were amplified with PCR (PyroMark PCR Kit, Qiagen) in the bisulfite-modified DNA according to the manufacturer’s protocols. PCR products were pyrosequenced on a Pyromark ID with Pyrogold reagents (Biotage, Uppsala, Sweden) and methylation levels were analyzed with Pyro Q-CpG SW (Biotage). A sample of pooled DNA was run in each batch as internal control, which was used to calculate relative methylation levels. These relative methylation levels were used for all further analyses.