Renilla phrl tk control vector

The 1.7 kb and 0.6 kb 5’-upstream region of Fthl17 were amplified and sequenced. Each sequence was cloned into the CpG-free pCpGL-basic Luciferase vector [15 (link)] by ligation using TaKaRa DNA Ligation Kit (TaKaRa). Luciferase reporter constructs were either mock-treated or methylated in vitro with SssI CpG methyltransferase for 4 h at 37°C and purified with the QIAquick Purification Kit (QIAGEN). 500 ng of each reporter plasmid and 50 ng of Renilla phRL-TK control vector (Promega) were co-transfected into KLN205 cells and GS cells using Lipofectamine LTX (Invitrogen) and Neon Transfection System (Invitrogen), respectively. After 48h, cells were lysed. Then, the relative Luciferase activities were analyzed using the Dual-Luciferase Reporter Assay System (Promega) on a Lumat LB 9507 (Berthold). Firefly Luciferase activity of individual transfections was normalized against Renilla Luciferase activity.

The gene regions between 500 bp upstream and downstream from the TSSs of Hist1h2ba, Sycp1, and Taf7l were amplified and sequenced. The primer sequences are shown in Supplementary file 5. Each sequence was cloned into the CpG-free pCpGL-basic Luciferase vector (Klug and Rehli, 2006 (link)). Luciferase reporter constructs were either mock-treated or methylated in vitro with SssI CpG methyltransferase for 4 hr at 37°C and purified with the QIAquick Purification Kit (QIAGEN 28704). Reporter plasmid (500 ng) and Renilla phRL-TK control vector (50 ng; Promega E2241) were co-transfected into HEK293T cells cultured in DMEM containing 10% FBS using Lipofectamine LTX Reagent with PLUS Reagent (Invitrogen 15338100). After 48 hr, cells were lysed, and the relative luciferase activities were analyzed using the Dual-Luciferase Reporter Assay System (Promega E1910) on a Lumat LB 9507 (Berthold). Firefly luciferase (Luc) activity of individual transfections was normalized against Renilla luciferase activity. We analyzed data from three independent experiments to evaluate statistically significant differences.