Epitect 96 bisulfite kit

Complete bisulfite conversion and cleanup of the extracted DNA for methylation analysis were performed in a 96-well setup, using the EpiTect 96 Bisulfite Kit (Qiagen, Valencia, CA) according to manufacturer’s instructions for sodium bisulfite conversion of unmethylated cytosines in DNA isolated from FFPE tissue samples using a centrifuge. The samples were divided into 96-well plates for bisulfite conversion. For each sample, DNA was dissolved in a bisulfite mix and RNase-free water, in a total volume of 140 $\mu$ L using the provided EpiTect conversion plate. Bisulfite conversion was then performed using a thermocycler with a heated lid. Thermal cycler conditions consisted of an initial 5-minute denaturation step at 95 ${}^{\circ }$ C, a 25-minute incubation step at 60 ${}^{\circ }$ C, a 5-minute denaturation step at 95 ${}^{\circ }$ C, an 85-minute incubation step at 60 ${}^{\circ }$ C, a 5-minute denaturation step at 95 ${}^{\circ }$ C, a 175-minute incubation step at 60 ${}^{\circ }$ C, and finally an indefinite hold at 20 ${}^{\circ }$ C. The samples were then transferred to an EpiTect 96-plate for cleanup and elution using carrier RNA buffer, desalting buffer, de-sulfonating buffer, ethanol, and elution buffer. Multiple washing steps were performed before centrifugation at 40 ${}^{\circ }$ C to ensure the evaporation of residual ethanol and final elution.