All PCR products for luciferase reporter assays were ligated into Invitrogen’s pCR-Blunt (K270020) plasmid using T4 DNA Ligase (New England BioLabs, M0202S), at 40 C overnight. MIR196A enhancers and promoters were digested from pCR-Blunt and cloned into the luciferase reporter plasmid pGL3-Basic (Promega, E1751). Enhancers were cloned into the BamHI/SalI site whilst promoters were cloned into the multiple cloning site immediately upstream of the luciferase gene. See Supplementary Table 1 for primers.
MCF7 cells were transfected in antibiotic free media with 500 ng of modified pGL3 reporter constructs, 20 ng of pRL-TK (Renilla transfection control) and with 0.5 μL of Lipofectamine 3000 (Life Technologies, L3000-008). 48 h post transfection luciferase readings were measured using a DTX-880 luminometer and Dual-Glo Stop and Glo luciferase reporter kit (Promega, E2920), following the manufacturer’s recommended protocol.
MCF7 cells were transfected in antibiotic free media with 500 ng of modified pGL3 reporter constructs, 20 ng of pRL-TK (Renilla transfection control) and with 0.5 μL of Lipofectamine 3000 (Life Technologies, L3000-008). 48 h post transfection luciferase readings were measured using a DTX-880 luminometer and Dual-Glo Stop and Glo luciferase reporter kit (Promega, E2920), following the manufacturer’s recommended protocol.