GSM9206324: Healthy Control EM-Seq, Replicate 2; Homo sapiens; OTHER
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. For the cfRRBS technique, 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 using the NEBNext Multiplex Oligos for Illumina with the following conditions: 95 °C for 5 minutes, 16 cycles for 98 oC for 20 seconds, 65 oC for 15 seconds, 72 oC for 45 seconds, followed by a final extension at 72 oC for 5 minutes. The amplified libraries were then pruified using a 2.5x Ampure bead: library ratio. The purified DNA was assessed for quality using a Qubit 4 fluorometer (Thermofisher Scientific, USA) and BioAnalyser (Agilent, USA). For the EM-Seq technique, only the control DNA was sheared (i.e., CpG methylated pUC19 and unmethylated lambda DNA) to 300 bp using the Covaris M220 focused-ultrasonicator with 130 L microtube AFA Fibre pre-slit snap-cap tubes. Furthermore, a 2x bead purification protocol was used to purify the adaptor-ligated DNA, then 1.8x, 2x, and 1x bead purification protocols for the TET2-converted, deaminated, and amplified DNA, respectively. Each library was amplified using the following conditions: 98 oC for 30 seconds, 10 cycles for 98 oC for 10 seconds, 62 oC for 30 seconds, 65 oC for 60 seconds and a final extension at 65 oC for 5 minutes.