title: GSM8372311: Reference sample 100%; synthetic construct; Bisulfite-Seq
library_construction_protocol: Cell pellet samples were lysed based on the total cell count per sample and total volume received using M-digestion Buffer (ZymoResearch; Irvine, CA; cat# D5021-9) and 5-10μL of protease K (20mg/mL), with a final M-digestion concentration of 2X. The samples were incubated at 65°C for a minimum of 2 hours.20μL of the supernatant from the sample extracts were bisulfite modified using the EZ-96 DNA Methylation-Direct Kit™ (ZymoResearch; Irvine, CA; cat# D5023) as per the manufacturer's protocol with minor modification. The bisulfite modified DNA samples were eluted using M-elution buffer in 46μL.All bisulfite modified DNA samples were amplified using separate multiplex or simplex PCRs. PCRs included 0.5 units of HotStarTaq (Qiagen; Hilden, Germany; cat# 203205), 0.2μM primers, and 3μL of bisulfite-treated DNA in a 20μL reaction. All PCR products were verified using the Qiagen QIAxcel Advanced System (v1.0.6). Prior to library preparation, PCR products from the same sample were pooled and then purified using the QIAquick PCR Purification Kit columns or plates (cat# 28106 or 28183). ii. Recommended PCR cycling conditions: 95°C 15 min; 45 x (95°C 30s; Ta°C 30 s; 68°C 30 s); 68°C 5 min; 4°C ∞ iii. Customer samples were run alongside established reference DNA samples with a range of methylation. They were created by mixing high- and low-methylated DNA to obtain samples with 0, 5, 10, 25, 50, 75, and 100% methylation. The high-methylated DNA is in vitro enzymatically methylated genomic DNA with >85% methylation. The low-methylated DNA is chemically and enzymatically treated with <5% methylation. They are first tested on numerous gene-specific and global methylation assays using pyrosequencing. Libraries were prepared using a custom Library Preparation method created by EpigenDx. Next, library molecules were purified using Agencourt AMPure XP beads (Beckman Coulter; Brea, CA; cat# A63882). Barcoded samples were then pooled in an equimolar fashion before template preparation and enrichment were performed on the Ion Chef™ system using Ion 520™ & Ion 530™ ExT Chef reagents (Thermo Fisher; Waltham, MA; cat# A30670). Following this, enriched, template-positive library molecules were sequenced on the Ion S5™ sequencer using an Ion 530™ sequencing chip (cat# A27764).