GSM9079034: Non-malignant cfRRBS Patient #50; Homo sapiens; Bisulfite-Seq
cfDNA was extracted using Qiagen ccfDNA Midi Kit Cell-free DNA was extracted from 2-5 mLs of plasma using the QIAamp MinElute ccfDNA Midi Kit according to the manufacturer's instructions. cfDNA was quantified using the Qubit 4 fluorometer according to the Qubit dsDNAhigh-sensitivity assay kit protocol. An Agilent 2100 Bioanalyser instrument was used to assess the cfDNA quality to rule out possible genomic DNA contamination. 4-10 ng of cfDNA was dephosphorylated using recombinant shrimp alkaline phosphatase (rSAP). This was followed by endonuclease digestion suing the MspI restriction enzyme, then enr-repair and adenylation of the 3' ends. Followiing the ligation of custom adapters, exonucleases were used to degrade unligated and partially ligated DNA fragments. This was followed by the cleavage of adaptor hairpin using antarctic thermolabile UDG and endonuclease VII, thus generating single-stranded DNA required for bisultte conversion. The DNA was bisulfite-converted twice using the Epitect plus bisulfite kit. This was followed by PCR amplification and 2.5x Ampure bead purification. The purified DNA was assessed for quality using a Qubit 4 fluorometer (Thermofisher Scientific, USA) and BioAnalyser (Agilent, USA).