Methyleasy xceed

DNA was purified by phenol-chloroform extraction. Cells were lysed in lysis buffer (50 mM Tris-HCl (pH 7.8), 100 mM ethylenediaminetetraacetic acid (EDTA; pH 8.0), 100 mM NaCl, 1% SDS, and 500 μg/mL proteinase K) at 55 °C overnight. Bisulfite conversion was performed using the MethylEasy Xceed (Takara) according to the instructions provided by the supplier. The converted DNA was amplified by PCR using primers as described previously^{28 (link)}. PCR was performed using the TaKaRa EpiTaq HS (Takara) according to the protocol provided by the manufacturer. PCR products were cloned into bacteria using the TOPO TA Cloning Kit (Invitrogen). Eight clones for each sample were sequenced. The primer sequences that were used for PCR are listed in Supplementary Table 2.

Genomic DNA was isolated from pro-B(+), pro-B(–) and LMO2/pro-B(−) cells. The DNA was purified using NucleoSpin Tissue (TaKaRa Bio) and was treated with bisulfite using MethylEasy Xceed (TaKaRa Bio) according to the manufacturer’s instructions. For the evaluation of DNA methylation status of CpG island in Tcf7 promoter region, which contains 25 potential CpG methylation sites, bisulfite-converted DNA was amplified by PCR using TaKaRa EpiTaq HS (for bisulfite-treated DNA, TaKaRa Bio) and the following primer set: 5’-ttaagtttttattggtgaatgagtt-3’ and 5’-aaaaaactccaaaaataaaacccac-3’. Amplified PCR products were cloned into pMD20-T vector using Mighty TA-cloning kit (TaKaRa Bio) and then sequenced.

The methylation-specific polymerase chain reaction (MSP) was used to distinguish between hmMGMT and non-hmMGMT. Briefly, DNA samples extracted from tumor tissues were treated with bisulfite using MethylEasy Xceed (Takara Bio Inc.) according to the manufacturer’s protocol. Subsequently, MSP was carried out using the EpiScope MSP kit (Takara Bio Inc.). The primers used for PCR were described previously [25 (link)]. PCR was carried out in a 50-μL volume containing 4 μg of bisulfite-treated DNA, 25 μL of 2xMSP buffer, 1.2 μL of MSP enzyme, 0.5 μL of 100xSYBR Green I, 16.3 μL of nuclease-free water, and 1.5 μL of each of the two primers. The reaction was incubated at 95 °C for 30 sec, followed by 35 cycles at 98 °C for 5 sec, 55 °C for 30 sec, and 72 °C for 1 min, with a final incubation step at 16 °C.