Validation of hMeDIP on the SX-8G IP-Star® Automated System
Hydroxymethylated DNA Immunoprecipitation (hMeDIP) was performed using the hMeDIP kit (rat monoclonal antibody)(Cat. No. C02010031) and carried out on the SX-8G IP-Star® Automated System. The Rat IgG isotype antibody was used as negative control. 1 µg of DNA from mouse ES cells was prepared and sonicated with the Bioruptor® to obtain DNA fragments of 300-500 bp. Unmethylated, methylated and hydroxymethylated spike DNA controls (included in the Auto hMeDIP kit) were used in the IP reaction together with the genomic sheared DNA samples. Enrichments were assessed by qPCR using specific primer pairs for the unmethylated, methylated and hydroxymethylated DNA sequences. Sfi1 is a gene that has been identified as being hydroxymethylated using hMeDIP-seq. Human DNA from U2OS was used as negative control DNA.
These results show clearly that hydroxymethylated DNA is specifically immunoprecipitated (hydroxymethylated control and Sfi1) and validate therefore the hMeDIP assay based on Diagenode’s hMeDIP kit carried out on the SX-8G IP-Star Automated System.