GSM1981393: LNCaP_NOMe_EtOH_1; Homo sapiens; Bisulfite-Seq
Cells were trypsinized and centrifuged for 7 mins at 1000rpm, then washed in ice-cold PBS and resuspended in 1mL ice-cold Nuclei Buffer (10mM Tris,pH 7.4, 10mM NaCl, 3mM MgCl2, 0.1mM EDTA and 1% IGEPAL, plus protease inhibitors) per 5x106 cells and incubated on ice for 10 min. To obtain the nuclei, cells were dounce-homogenized for 15 times and incubated on ice for about 45 min, until single nuclei were visualized under the microscope. Nuclei were recovered by centrifugation at 3000 rpm for 5 min and washed in Nuclei Wash Buffer (10mM Tris,pH 7.4, 10mM NaCl, 3mM MgCl2 and 0.1mM EDTA containing protease inhibitors). Freshly prepared nuclei (2x105 cells) were resuspended in 1X M.CviPI reaction buffer (NEB), then treated with 150U of M.CviPI (NEB; 50,000U/mL) in 15µL 10X reaction buffer, 45µL 1M sucrose and 0.75µL SAM in a volume of 150uL. Reactions were quenched by the addition of an equal volume of Stop Solution (20mM Tris-HCl [pH 7.9], 600mM NaCl, 1% SDS, 10mM EDTA, 400µg/ml Proteinase K) and incubated at 55°C overnight. DNA was purified by phenol/chloroform extraction and ethanol precipitation. 50ng of genomic DNA was bisulfite converted using the EZ DNA Methylation Gold kit (Zymo Research, Cat: D5005) following the manufacturer’s protocol. The bisulfite converted DNA was then subjected to library preparation using the EpiCenter kit (now known as the EpiGnome library preparation kit or TruSeq DNA Methylation kit, Illumina, Cat: EGMK81312). In this “post bisulfite conversion” library preparation method, single stranded bisulfite converted DNA was randomly primed to synthesize DNA strands with a specific sequence tag followed by 3’ tagging and PCR amplification using the FailSafe PCR enzyme (Gene Target Solutions,Cat: FSE51100). The PCR was performed for 10 cycles followed by purification using Agencourt AMPure XP beads (Beckman Coulter, Cat: A63881). The purified library was eluted in a final volume of 20ul pure water. Quality of the library obtained was checked using the DNA High sensitivity chip on the Agilent Bioanalyzer. The library was quantified using Qubit and KAPA Biosystems Library quantification kit according to manufacturer’s instructions.
GEO Accession
GSM1981393