Dual glo reporter assay system

The dual-luciferase vectors psiCHECK-SOCS3–3’UTR-WT and psiCHECK-rcmiR −222-WT were constructed by synthesizing the candidate seed sequences in the 3′-UTR of SOCS3 or the reverse complementary sequence of miR-222 and inserting them into the psiCHECK-2 vector. For mutant vectors, 3–4-bp mutations were introduced into the seed sequences. All plasmids were confirmed by DNA sequencing. For reporter assays, HEK-293 T cells were seeded in 24-well plates and transfected with 0.8 μg recombinant vectors alone or plus 30 nM mimics or inhibitors. Firefly and Renilla luciferase activities in cell lysates were measured 48 h later using the dual-Glo reporter assay system (Promega).

HEK293T cells were co-transfected with Sp1-overexpressing plasmid (OriGene, Rockville, MD, USA) or empty vector and the reporter plasmid for DNA-PKcs (PRKDC) promoter [Ppro-RB-Report-PRKDC-WT (−1000~−1)/Mut (−78~−55)] or corresponding empty plasmid (RiboBio, Guangzhou, China) using Lipofectamine^® 3000 (Thermo Invitrogen, Carlsbad, CA, USA). Dual luciferase assay was performed after 48 h using the Dual-Glo^® Reporter Assay System (Promega Biotech Co., Beijing, China) according to the manufacturer’s protocol. Luciferase activity was measured as the ratio of the firefly luciferase signal to the Renilla luciferase signal. All measurements were normalized to the control group, and the experiment was performed in triplicate.

For experimental validation of the enhancer function of TEs in mouse ESCs, we used the RW4 cell line, and cultured it as previously described66 (link). RW4 cells were cultured in 0.1% gelatin-coated Petri dishes in culture medium containing DMEM (Invitrogen) supplemented with 10% fetal bovine serum (Gibco), 10% neonatal calf serum (Hyclone), nucleosides (Sigma), 1,000 U ml⁻¹ leukemia-inhibiting factor (Sigma) and 0.1 mM β-mercaptoethanol (Gibco).
To estimate the regulatory potential of TE and non-TE sequences in mouse ESCs, we used the Dual-Glo luciferase assay (Promega). For the luciferase assay, we cloned TEs into the pGl4.23 vector (containing firefly luciferase; Supplementary Tables 5A–C and 9). We co-transfected the TE-plasmid with pRL-TK (containing renilla luciferase) using lipofectamine (X-GENE-HD at 1 μl for 1 μg of plasmid DNA) into RW4 cells plated in 0.1% gelatin-coated 96-well plate. After 24 h, we assayed the luciferase levels according to the Dual-Glo reporter assay system (Promega).
We performed each luciferase experiment in triplicate, and repeated each experiment three times. The luciferase fold change is estimated as the ratio of firefly and renilla luciferase for each TE, and then normalized by the empty vector (pGl4.23 with no insert).

Wild‐type SMAD7 3′UTR was purchased from OriGene (Clone NM_005904). Mutant SMAD7 3′UTR was obtained by PCR mutagenesis of the wild‐type SMAD7 3′UTR, followed by sequencing for quality control (Fig 3B). Mycoplasm‐free (MycoAlert, Lonza) human embryonic kidney (HEK) 293 cells (gift from Prof. W. Herr, University of Lausanne) were maintained in DMEM + 10% FBS. HEK 293 cells were transfected (Lipofectamine RNAimax) with pRL for standardization (Renilla luciferase reporter under CMV promoter, 0.625 ng/ml), miR‐21‐3p mimic or scrambled sequence (miRIDIAN, Thermo Scientific Dharmacon, 50 nM), and Firefly luciferase reporter—wild‐type or mutant Smad7 3′UTR reporter (40 ng/ml). Cells were harvested 24 h after transfection for luciferase assay analysis (Dual‐Glo^® Reporter Assay System, Promega) using the Microplate luminometer GloMax^®‐9 (Promega).

The dual-luciferase vectors were constructed by synthesizing the seed sequences of the 3'-UTR of TPT1 or the reverse complementary sequence of miR-216a and inserting these into psiCHECK-2 vectors. The corresponding mutant vectors were constructed by introducing 3-bp mutations into the above seed sequences. To verify the specific targeting of TPT1 by miR-216a-5p, HEK-293 T cells were seeded in 24-well plates and transfected with 0.8 μg of recombinant vectors, either alone or with miR-216a-5p mimic or inhibitor. Firefly and Renilla luciferase activities in cell lysates was measured 24 h later using the dual-Glo reporter assay system (Promega, Madison, USA).

Sensor and mutant vectors for Tgfbr2 were created from the psiCHECK-2 vector (Promega). Each of the control and sub-cluster vectors, and each of the sensor and mutant vectors were co-transfected into HEK293T cells using Lipofectamine LTX (Thermo Fisher Scientific) and cultured for 24 h. Renilla luciferase–firefly luciferase ratios in the cells were measured using a Dual-Glo reporter assay system (Promega) according to manufacturer instructions.