Figure 1
Workflow of stem cell dynamics assay. The assay is performed on archival paraffin embedded (FFPE) material from normal-looking tissues. Single crypts are laser microdissected, and DNA is isolated. Next, the extracted genomic DNA is treated with sodium bisulphite to convert unmethylated cytosines into uracil; methylated cytosines are protected from this turnover. A nested PCR is performed to target the CSX sequence. The PCR products are extended with Iontorrent PGM sequencing adapters and sequenced following the manufacturers protocol. The various methylation patterns per crypt can then be clustered by adapters. Different numbers of reads are found for each pattern including PCR errors which occurred below 1% count. Meanwhile, as a technical control, the same PCR products are cloned into bacterial vectors and enumerated after a sequence analysis of cloned inserts. 10 clones were selected for sequencing so for each crypt, the total number of counts are 10. CSX, cardiac-specific homeobox. Different colors represent different methylation patterns.
