GSM8464478: Plasma, BorderlineRejection, SOT0032-1-1; Homo sapiens; Bisulfite-Seq
Cell-free DNA was isolated from plasma specimens using the QIAMP CAN Kit per manufacturer's instructions. In total of 5 ng of DNA was aliquoted from each sample. Unmethylated λDNA was added to each sample at 0.5% w/v. The samples were then concentrated with a SpeedVac to a volume of 20µL. Due to the fragmented nature of cfDNA, further fragmentation of the samples was not required. The samples then underwent bisulfite conversion with an EZ DNA Methylation- Gold kit (Zymo, Cat. No. D5006). The samples were eluted off the spin columns with 15μl of low EDTA TE buffer (Swift, Cat. No. 30024) before library preparation.  Low-input libraries were prepared using an ACCEL-NGS Methyl-Seq Library kit (Swift, Cat. No. 30024) with a Methyl-Seq Set A Indexing Kit (Swift, Cat. No. 36024), following the protocol associated with the kit. Bead cleanup steps were performed with SPRIselect beads (Beckman Coulter, Cat. No. B23318). Six PCR cycles were performed to amplify the samples. Final libraries were quantified with a Qubit dsDNA HS Assay Kit and size was determined using a BioAnalyzer High Sensitivity DNA Kit (Agilent, Cat. No. 5067-4626). The libraries were then sequenced on the Illumina NovaSeq6000 System using 150bp paired-end sequencing.