Pcpgf promlc

A DNA sequence spanning an intron 1 region of CTSH (chr15: 79,236,578–79,237,097, hg19) was cloned into the intron of the pCpG-free-lucia plasmid (Invivogen; catalog no.: pcpgf-promlc), which was denoted as pCpG-free-CTSH intron 1-lucia plasmid thereafter. The expression of the lucia gene was driven by a modified human EF-1α promoter. The modified EF-1α promoter in the pCpG-free-lucia plasmid was devoid of CpG sites and therefore was ideal for this experiment because it allowed us to only methylate the CTSH intron 1 without getting methylated itself. In contrast, the CTSH promoter is enriched with CpG sites. CTSH promoter would be methylated when the plasmid is in vitro methylated and therefore could not be used for this experiment. To truncate CpGs within this region, site-directed mutagenesis primers were designed using the NEBaseChanger tool (New England Biolabs). Site-directed mutagenesis was performed using the Q5 site-directed mutagenesis kit (New England Biolabs; catalog no.: E0554S) following the manufacturer's protocol. pCpG-free-CTSH intron 1-lucia plasmids were transformed into the ChemiComp GT115 E. coli cells (Invivogen; catalog no.: gt115-11). Selected colonies were grown overnight for DNA extraction.