GSM5602630: Dam_Met4_2; Homo sapiens; OTHER
Genomic DNA (gDNA) extraction was performed using a DNeasy Blood & Tissue Kit (Qiagen) according to manufacturer instructions For methylated GATC library preparation, gDNA (300 ng) was digested using DpnI (New England Biolabs) for 6 h at 37°C, followed by DpnI inactivation for 20 min at 80°C. dsAdR adaptor ligation was performed using T4 DNA ligase (New England Biolabs) overnight at 16°C, followed by ligase inactivation for 10 min at 65°C. Digestion using DpnII (New England Biolabs) was then performed for 1 h at 37°C, followed by DpnII inactivation for 20 min at 65°C. The reaction mixture was purified using a PCR purification kit (Qiagen) and eluted in RNase-free water. Initial PCR amplification was performed using AdR_PCR primers and SYBR Select master mix (PCR cycles 1–5), with the extension temperature was set at 72°C. The optimum number of remaining cycles was determined by plotting linear Rn (passive reference dye-normalised reporter signal) versus cycle number and calculating the number of cycles corresponding to 10× initial fluorescence intensity to avoid PCR duplication. Methylated GATC gDNA fragments were purified using solid-phase reversible immobilisation beads and eluted in RNase-free water. GATC gDNA fragments (50 ng) were treated with Nextera Tn5 transposase (Illumina) for 5 min at 37°C with shaking. The transposase reaction was stopped by adding binding buffer from the PCR purification kit, and tagged DNA fragments were eluted in RNase-free water. DNA fragments were indexed and barcoded and PCR-amplified using Ad1_noMX and Ad2.n primers and SYBR Select master mix, with initial holding temperature of 72°C (5 min) followed by 98°C (30 s). The optimum number of cycles was determined by qPCR (as above) at a denaturing temperature of 98°C (10 s), annealing temperature of 63°C (30 s) and extension temperature of 72°C (1 min). Library fragments were purified using solid-phase reversible immobilisation beads and eluted in RNase-free water