Gblock

Luciferase reporter constructs were engineered by cloning the WT NOD1 3’-UTR, a NOD1 3’-UTR with the miR-15b/16 binding site mutated (miR15b/16-mut), or NOD1 3’-UTR with miR-15b/16 and miR-106a binding sites mutated (miR-mut) (gBLOCK, IDT) into the pGL3 vector (Promega). For luciferase assays, HEK-293 cells were seeded at 4×10⁴ cells/well in a 48-well plate overnight. Cells were transfected with 200 ng NOD1 WT or miR-15b/16-mut 3’-UTR luciferase reporter constructs and 10 ng eGFP using FuGENE HD transfection reagent (Promega). Where noted cells were co-transfected with either miRNA mimics, LNA inhibitors or TSB at a final concentration of 100 nM using HiPerfect (Invitrogen) for 36–48 h. Cells were washed with PBS and lysed in cell lysis buffer (9803, Cell Signaling Technology). Luciferase activity was measured with the Steady-Glo Luciferase Assay System (Promega) using a microplate reader (Biotek Synergy H4 Plate reader). Luciferase activity was normalized for transfection efficiency (eGFP) and plotted as a fold change over the respective control.

To validate the expression values obtained by MPRAu, we selected 18 oligos consisting of nine ref/alt pairs. Five of the oligos were selected as no-skew controls for having uncorrected p-values of greater than 0.01. We designed the same 101 bp sequence that was tested by MPRAu as a gBlock (IDT) and cloned each into the pmirGLO dual-luciferase reporter vector (Promega, E1330). Cells were plated in a 96 well plate and grown to a density of 80%-90%, then transfected with a mixture of 0.2 μL Lipofectamine 2000, 500 pg of the cloned dual-luciferase vector, and 49.5 ng of pGL4.23, a promoterless control vector (Promega, E8411). We performed six transfection replicates per oligo (all on the same 96-well plate). Cells were incubated with transfection reagents for 24 hours, monitored by fluorescent microscopy, and then split 1:3 into a new 96-well plate. After 24 hours (48 hours post-transfection), firefly and Renilla luminescence were read from each well using the Dual-Glo Luciferase Assay (Promega, E2920). Firefly luciferase luminescence for each well was normalized to the Renilla luciferase luminescence for the same well, and each experiment was normalized as a log-ratio value relative to the mean of a control oligo with an MPRAu RNA/DNA ratio of −0.2 (Supplementary Table 2).