GSM9023163: hNPC(AG09429), input sample, MG treatment, 24h(GlyDIP); Homo sapiens; OTHER
Cells were collected from 10 cm dishes at ~80% confluency, washed twice with ice-cold PBS, and pelleted by centrifugation at 300 g for 5 minutes at 4°C. Cell pellets were resuspended in GenDNA Digestion Buffer containing 0.75 µL Proteinase K and 7.5 µL of 200 mM D-P (final 10 mM) in 150 µL total volume, and incubated at 50°C for 12–18 hours with shaking at 300 rpm. DNA was purified using phenol:chloroform:isoamyl alcohol (25:24:1) extraction, followed by chloroform cleanup and ethanol precipitation. DNA pellets were washed with 70% ethanol and dissolved in 15 µL TE buffer. Samples were incubated at 37°C for 3 hours with shaking and stored at –20°C until further processing. Libraries were prepared using the NEBNext Ultra II DNA Library Prep workflow (New England Biolabs; Cat. Nos. E7546L, E7595L, E7600S), starting from 2 μg of fragmented genomic DNA. Libraries were ligated with NEBNext adaptors, USER-treated, and PCR-amplified according to the manufacturer's protocol. Final libraries were sequenced using 150 bp paired-end reads on the NovaSeq X platform.