GSM2090574: NC-P-19; Homo sapiens; Bisulfite-Seq
Plasma from patients were processed to extract circulating DNA. The DNA extracted from plasma were then concentrated using ethanol precipitation and eluted in 15 uL nuclease-free water. 1-1.5 ng of DNA were digested with 10 U of MspI (Thermoscientific), 1X Tango buffer (Thermoscientific), and 10 pg of unmethylated lambda DNA (New England Biolabs) as control for ~13 h at 37 ˚C, then heat inactivated at 65 ˚C for 20 min. Next, 5 U of Klenow fragment, exo- (Thermoscientifc) and a mixture of dATP, dGTP, and dCTP (New England Biolabs) were added for a final concentration of 1 mM, 0.1 mM, and 0.1 mM for dATP, dGTP, and dCTP respectively. The mixture was gently vortexed, and incubated at 30 ˚C for 20 min, 37 ˚C for 20 min, and finally 75 ˚C for 10 min. To perform adaptor ligation, the dA-tailed DNA were added to a 5 uL mixture of 1X Tango buffer, 30 U of T4 DNA Ligase, HC (Thermoscientific), 2.5 mM ATP, and 500 nM individual TruSeq multiplexing methylated adaptors. The combined mixture was gently vortexed, incubated at 16 ˚C for ~20 h, then heat inactivated at 65 ˚C for 20 min. The ligation mixture was purified using Agencourt AMPure XP beads (Beckman Coulter), and eluted in 20 uL of nuclease-free water. The ligated products were then bisulfite converted using the MethylCode(™) Bisulfite Conversion kit (Life Technologies). Two rounds of amplification were performed after bisulfite conversion. The first round was using PfuTurboCX (Agilent Technologies) for 12 cycles in 50 uL total volume, then the second round was performed using Phusion HotStart Flex (New England Biolabs) master mix for 9 cycles in 50 uL total volume. Final PCR products were purified, pooled in equimolar ratios, and size selected using polyacrylamide gels for 150-400 bp. Libraries were sequenced on both MiSeq and HiSeq RapidRun for PE 100 cycles.
GEO Accession
GSM2090574