GSM9470645: R342C_ID; Mus musculus; Bisulfite-Seq
Nuclei were extracted using cold lysis buffer (10 mM Tris-HCl pH 7.4, 10 mM NaCl, 0.5 mM spermidine, 1.5 mM MgCl2, 0.1 mM EDTA, 0.25% IGEPAL CA-630) for 3-6 minutes after checking nuclei under a light microscope. The sample was centrifuged for 5 minutes at 800x g at 4°C, washed with 1 ml ice-cold nuclei wash buffer (10 mM Tris-HCl pH 7.4, 50 mM NaCl, 0.5 mM spermidine, 1.5 mM MgCl2, 0.1 mM EDTA), and centrifuged again for 5 minutes at 800x g at 4°C. The nuclei were resuspended in 564 µL 1X GpC MTase reaction buffer and equilibrated to 37°C. This was followed by 6 minutes of enzyme treatment (300 µL 1 M sucrose, 34 µL 10X GpC methyltransferase reaction buffer, 3 µL 32 mM S-Adenosylmethionine (SAM) (NEB, B9003), 100 µL GpC methyltransferase (M.CviPI), followed by another 6 minutes of enzyme treatment (50 µL GpC methyltransferase (M.CviPI), 3 µL 32 mM S-Adenosylmethionine (SAM)). An equal volume of pre-warmed 2X stop buffer (20 mM Tris-HCl pH 7.4, 600 mM NaCl, 10 mM EDTA, 1% SDS) was added and mixed well. Proteinase K (200 µg/mL) was added to the GpC methylation reaction and incubated for 4 hours at 56°C, followed by RNase A (100 µg/mL) for 30 minutes at 37°C, followed by an additional round of proteinase K (200 µg/mL) for 30 minutes at 37°C. HMW DNA was extracted using phenol-chloroform and precipitated by isopropanol. Briefly, an equal volume of phenol-chloroform was added to the samples and mixed vigorously. The samples were centrifuged at 13,000 rpm for 10 minutes at room temperature. The aqueous phase was transferred to a clean tube, and 0.7-1 times the volume of isopropanol was added, mixed well, and incubated at room temperature for 30 minutes. The samples were then centrifuged at 13,000 rpm for 10 minutes at 4°C. The DNA pellet was washed with 1 ml pre-chilled 70% ethanol. After removing all the ethanol, the DNA was dissolved in EB buffer and incubated at 37°C for 30 minutes. The DNA was fragmented prior to bisulfite conversion. Fragmentation was performed via sonication (Diagenode Bioruptor) to produce 350 bp fragments. Bisulfite conversion was performed using the EZ DNA Methylation-Gold Kit, and the library was prepared using the xGen TM Methyl-Seq DNA library prep kit following the manufacturer's protocols.