MluI R6381

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	The wild-type and mutant CLOCK 3×RORE oligos were digested with XhoI and MluI (Promega). The pTL-Luciferase vector was also digested with XhoI and MluI (Promega).	All fragments were gel purified and ligated using T4 DNA Ligase (Promega). The constructs were verified by sequencing.

Protocol tips
The wild-type and mutant CLOCK 3×RORE oligos were digested with XhoI and MluI (Promega). The pTL-Luciferase vector was also digested with XhoI and MluI (Promega).
Downstream tips
All fragments were gel purified and ligated using T4 DNA Ligase (Promega). The constructs were verified by sequencing.

Publication protocol

The putative CLOCK RevRE and a mutated RevRE were synthesized as a three-times repeat (3×RevRE) with XhoI and MluI restriction sites on the ends (IDT). The wild-type and mutant CLOCK 3×RORE oligos were digested with XhoI and MluI (Promega). The pTL-Luciferase vector was also digested with XhoI and MluI (Promega). All fragments were gel purified and ligated using T4 DNA Ligase (Promega). The constructs were verified by sequencing. The p3x-FLAG-REV-ERBα has already been described previously [13]. Human HepG2 cells were maintained and propagated in minimum essential medium supplemented with 10% fetal bovine serum at 37°C under 5% CO2. For transfections, HepG2 cells were plated in 96-well plates at a density of 15×103 cells/well 24 h prior to transfection. Transfections were performed using Lipofectamine 2000, according to manufacturer's instructions (Invitrogen). Per well, the transfection mixture included 50 ng Renilla luciferase as an internal control, 100 ng of the appropriate CLOCK luciferase construct, and 100 ng of the p3x-FLAG-REV-ERBα expression construct. The luciferase activity was measured using the Dual-Glo luciferase assay system 24 h after transfection (Promega). The luciferase readings were normalized by well to the Renilla readings. For treatment with GSK4112, HepG2 cells were plated into 12-well plates. Cells were then treated with 10 µM GSK4112 or equivalent volume of vehicle. Treatment lasted for 24 hrs. Cells were harvested for RT-PCR determination of CLOCK expression.

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