Data Supplement Title: Circulating methylated SEPT9 DNA in Plasma is a Biomarker for Colorectal Cancer. Theo deVos1, Reimo Tetzner2, Fabian Model1,3, Gunter Weiss2, Matthias Schuster2, Jü rgen Distler2, Kathryn V. Steiger1 , Robert Grü tzmann5, Christian Pilarsky5, Jens K. Habermann6, Phillip R. Fleshner7, Benton M. Oubre8, Robert Day1, Andrew Z. Sledziewski1, Catherine Lofton-Day1 1) Detailed mSEPT9 Assay Protocol: DNA Extraction: DNA was extracted from 5 mL of blood plasma using a modified viral DNA/RNA extraction kit (chemagen AG, Baesweiler Germany). Plasma samples were thawed at room temperature and extracted following the directions of the kit with modifications. Samples were lysed and treated with protease at 56C for 10 min in a 50 mL Falcon tube. 100 L of magnetic particles and 15 mL of binding buffer were then added, and binding was performed for 60 min at room temperature on a rotator. Magnetic particles were captured for 4 min, the supernatant discarded and the pellet was resuspended in 3 mL of wash buffer. 1.5 mL of particle solution were transferred to a 2 mL SafeLock, the beads captured and the supernatant discarded. This was repeated to complete the 3 mL transfer. Tubes were briefly centrifuged and the residual wash buffer was removed by pipetting after bead separation. The tubes were then placed in a 56C dry block for 5 min, 100 l of elution buffer was added, the tubes incubated at 65C with shaking on a thermomixer for 15 min, the particles separated on a magnetic stand and the eluted DNA transferred to a 0.5 mL SafeLock tube (Eppendorf). A 5l aliquot of the DNA sample was transferred to 45 l of elution buffer for the measurement of genomic DNA. Bisulfite Conversion: T...
