GSM6466267: sDMEM 15 min, biol rep1; Homo sapiens; OTHER
After labeling, genomic DNA was isolated using a PureLink Genomic DNA mini kit. 2 µg genomic DNA was then diluted in 100 µL 1× PBS supplemented with 25 mM K3BO3 and 20 mM DBCO-PEG4-biotin. The mixture was shaken at 37 °C for 1.5 h, after which 5 µL RNase A was added, followed by another 5 min incubation at 37 °C. DNA was then purified from the reaction by using the DNA Clean & Concentrator-5 kit and was fragmented to 150-350 bp by sonicating 30 cycles at 30s-on/30s-off setting using Diagenode Bioruptor Pico. 5% of the sonicated DNA was set aside and saved, and the rest was used for enrichment with 10 µL Dynabeads MyOne Streptavidin C1. The beads were washed with three times with 1× B&W buffer (5 mM Tris-HCl pH 7.4, 0.5 mM EDTA, 1 M NaCl, 0.05% Tween-20), resuspended in 95 µL 2× B&W buffer, and mixed with sonicated DNA. Binding was performed at room temperature for 15 min. The beads were then washed 5 times in 1× B&W buffer and were subsequently resuspended in 15 μL H2O. Beads were then heated at 95 °C for 10 min to elute DNA; DNA eluate solution was then moved onto ice for library construction. Sonicated/sheared enriched DNA libraries were constructed using a Accel-NGS Methyl-Seq DNA library kit