GSM4626846: R1 ESC; Mus musculus; Bisulfite-Seq
Cell pellets from trypsinized R1 and Tet/Tet ESCs were resuspended in 1X PBS and centrifuged at 1,000 rpm for five min at room temperature in a swing-out rotor. The pellets were washed once again with 1X PBS, resuspended in a lysis buffer (10 mM Tris-HCl pH 8.0, 5 mM EDTA, 50 mM NaCl, 0.5% SDS) and treated with Proteinase K (50µg/ml) at 50℃ for two hours. The cell lysates were extracted with phenol: chloroform (1:1 ratio), centrifuged at 12,000 rpm for 10 min at room temperature and the supernatants were extracted with chloroform and centrifuged in the same manner. DNAs in the supernatants were precipitated using sodium acetate (final concentration = 0.3 M, pH 5.2) and an equal volume of isopropanol for five minutes by gentle inversions. The precipitated DNAs were spooled using sterile glass rods, washed with 70% ethanol, dried at room temperature for 15-30 minutes and dissolved in 1X TE buffer (10mM Tris-Cl, 1mM EDTA, pH 8.0). In case of neurons, confluent dishes of neurons were scraped with lysis buffer(RLT buffer with BME) and further QIAGEN All Prep Kit(Cat No. 80204) procedure was followed for the DNA extraction. The extracted DNAs were then quantified using NanoDrop. 200 ng of starting input genomic DNA was digested with 30 units of MspI (NEB) and then purified with Zymo Research DNA Clean & Concentrator™-5 (Cat#: D4003). Fragments were ligated to pre-annealed adapters containing 5'-methyl-cytosine instead of cytosine according to Illumina's specified guidelines. Adaptor-ligated fragments ≥50 bp in size were recovered using the DNA Clean & Concentrator™-5 (Cat#: D4003). The fragments were then bisulfite-treated using the EZ DNA Methylation-Lightning™ Kit (Cat#: D5030). Preparative-scale PCR was performed and the resulting products were purified with DNA Clean & Concentrator™-5 (Cat#: D4003) for sequencing on an Illumina HiSeq.
GEO Accession
GSM4626846