GSM8683161: NPC, HT_2, rrbs-seq; Homo sapiens; Bisulfite-Seq
Tissue or PBS-washed cells were lysed in a buffer containing 100 mM NaCl, 10 mM Tris-HCl (pH 8.0), 25 mM EDTA (pH 8.0), 0.5% SDS, and 200 µg/mL Proteinase K for 2 hours at 50°C. An equal volume of Phenol : Chloroform : Isoamyl alcohol (25:24:1) (Cat. No. P1021) was added to the lysate, and the supernatant was retained after centrifugation. This was mixed with twice the volume of pre-chilled ethanol and incubated at -20°C for 10 minutes to precipitate the DNA. After centrifugation at 13,000 rpm for 15 minutes at 4°C, the pellet was washed twice with pre-chilled 70% ethanol. The DNA was then dissolved in 10 mM Tris-HCl (pH 8.0). 10 ug of DNA were sonicated to approximately 150 bp fragments using the Covaris S220, followed by end repair and adenylation. Fragments were ligated with custom hairpin and sequence adapters. Enrichment for CG-rich fragments was performed using Msel (T/TAA) and Mluel (/AATT) restriction enzymes. Dynabeads M-280 Streptavidin were utilized to isolate fragments with successfully ligated hairpin adapters. Bisulfite conversion was conducted with the Epitect Plus DNA Bisulfite Kit following a specified temperature and time protocol (95℃ for 5min, 60℃ for 26min, 95℃ for 5min, 60℃ for 60min, 95℃ for 5min, 60℃ for 1h 25min, 95℃ for 5min, 60℃ for 2h 55min). Library concentrations were quantified using the Qubit 4.0 fluorometer. Amplification and indexing were performed using KAPA HiFi Hotstart Uracil+ReadyMix. The final libraries were size-selected for 350-550 bp fragments and subjected to paired-end sequencing on the NovaSeq 6000 platform