Top10 chemically competent cells

TERT promoter regions were cloned into a pNL1.1 vector (Promega), transformed in TOP10 chemically competent cells (Lifetech), and verified by Sanger sequencing. To generate methylated versus unmethylated plasmids, 1 μg of plasmid was treated with SssI (NEB) or mock as per manufacturer’s instructions. To ensure complete methylation, plasmid DNA was treated with SssI twice. Cells in culture were seeded into 96 well plates and transfected in suspension using X-tremeGENE HP DNA Transfection Reagent (Roche). Transfection complexes were prepared such that each reaction contained 11 μl Opti-MEM, 59 ng empty pCR2.1-TOPO vector, 5 ng pGL4.53(luc2/PGK) vector (Promega), 1 ng pNL plasmid, and 0.12 μl HP transfection reagent. After 2 days, transfected cells were lysed in 50 ul passive lysis buffer (Promega), transferred to black 96 well plates, and measured for reporter activity by mixing 50 μl of ONE-Glo EX Luciferase Reagent and NanoDLR Stop & Glo Reagent sequentially as described in the Nano-Glo Dual Luciferase Assay System manual (Promega).

DNAs from cells were extracted using DNeasy (QIAgen). DNA (100 ng) was bisulfite converted using an EZ DNA Methylation Gold Kit (Zymo). Bisulfite converted DNA was eluted in 30 μl water, of which 10 μl was used for a 40 μl PCR reaction containing 1x Buffer, 1.5 mM MgCl₂, 250 nM each dNTPs, 12.5 μg BSA, 6.25% DMSO, 5 units of Platinum Taq (Lifetech), 400 nM of forward and reverse primers (TertMut_BSF2_forward 5′-GAAAGGAAGGGGAGGGGTTGGGAGGG and TertMut_BSF2_reverse 5’-CCTCCACATCATAACCCCTCCCT) and 5 units of Platinum Taq (Lifetech). The following cycling conditions were used: 1 cycle of 95 °C for 3 min; 35 cycles of 95 °C for 30 s, 56 °C for 30 s, and 72 °C for 1 min; and 1 cycle of 72 °C for 5 min. Products were run on a 2% agarose gel, excised and cloned into the pCR2.1-TOPO vector system (Lifetech). The vectors were transformed in TOP10 chemically competent cells (Lifetech). Plasmid DNA was purified from colonies using a Plasmid Purification Kit (Qiagen), and subsequently analyzed by Sanger sequencing. Sanger bisulfite sequencing data were analyzed using a custom Java program called DNAMethylMap, which facilitates the analysis of Sanger bisulfite sequencing clones with virtually bisulfite converted reference amplicon sequences (B.K., S.Y., unpublished).