Miridian

For target validation, astrocytes were transfected with dual luciferase reporters (TIMP-2 and control clones, Cat # HmiT018093-MT06, and CmiT000001-MT06, respectively, GeneCopoeia™, Rockville, MD, USA) according to the manufacturer’s instructions. Following 48 h, cells were transfected with miRNA-301a-3p mimics (50 nM miRIDIAN, Dharmacon Inc., Lafayette, CO, USA) using the Dharmafect 4 transfection reagent. Luciferase assays were conducted 48 h after mimic treatment, using the Luc-Pair™ Duo-Luciferase HS Assay Kit (GeneCopoeia™), according to the manufacturer’s instructions. For functional evaluation of miR-301a-3p, astrocytes were treated with 50 nM miRNA-301a-3p mimics for 48 h, after which RNA was isolated for analysis.

We grew HeLa cells in Dulbecco's modified Eagle's medium (Lonza) with 10% FBS in 5% CO₂ at 37ºC and seeded them at a density of 1 × 10⁴ cells per well into 96-well plates. Cells were transfected in triplicate 24/48 h later with Lipofectamine 2000 (Invitrogen), 150 ng of a 3′UTR luciferase vector (see below), and 50 nmol of test miRNA mimic (miR-58, miR-80, miR-81 and miR-1834; miRIDIAN, Dharmacon) or the standard control miRNA mimic miR-67 provided by the manufacturer. The 3′UTRs (wild-type and mutant) of sma-6, dbl-1, daf-1, daf-7 and daf-4 were cloned after the renilla reporter gene in the vector psiCHECK-2 (Promega). For obtaining the 3′UTR mutants, we mutated the second, third and fourth positions of the predicted mir-58 family binding sites in 3′UTRs by PCR (primers in Supplementary Table S3). Dual-luciferase reporter assay were performed 48 h after transfection using Dual-Luciferease Reporter Assay System (Promega) to detect firefly and renilla luciferase activity, and luminescence was measured with an Infinite M200 TECAN luminometer (TECAN Group Ltd). Renilla luciferase activity was first normalized using the firefly luciferase activity as intraplasmid transfection reporter. Resulting values for miRNA-3′UTR co-expression were further normalized to those from the same 3′UTRs but incubated with control mimic miRNA (miR-67).

Human colon epithelial cell lines Caco-2 and CRL-1790 were purchased from American Type Culture Collection (ATCC). Cells were cultured in Eagle's Minimum Essential Medium (EMEM; ATCC) containing 20% (Caco-2) or 10% (CRL-1790) Fetal Bovine Serum (FBS; ThermoFisher) at 37°C and 5% CO₂. Expression of miR mimics in Caco-2 and CRL-1790 cells was carried out using DharmaFect transfection reagent (GE Dharmacon) according to the manufacturer's protocols. Mature miRNA mimic constructs (miRIDIAN™, GE Dharmacon) were dissolved in siRNA buffer (GE Dharmacon) to achieve final concentrations of 100 nM. For RNA isolation from transfected CRL-1790 cells, transfection media containing miR mimics or vehicles (siRNA buffer) were replaced with fresh feeding media at 24 hours posttransfection and cells were incubated for an additional 24 hours. Total RNAs were isolated using a microRNA isolation kit (Qiagen). The C.elegans cel-miR-67 mimic (GE Dharmacon) was used as a nonmammalian miR control.

Dgcr8-knockout and WT parental cells were previously derived in the Blelloch lab (3 (link)ref. 3) and can be obtained from Novus biologicals. Cells were maintained in knockout DMEM medium (Invitrogen) supplemented with 15% fetal bovine serum, LIF and 2i (PD0325901 and CHIR99021) as per standard techniques32 (link). Cells were transfected with miRNA mimics (MIRIDIAN, GE Dharmacon) using Dharmafect 1 as previously described33 (link). A fluorescence tagged (Dy547) control mimic was used to evaluate transfection efficiency (Supplementary Fig. 1a). Flow cytometry (LSRII) and microscopic analysis of transfected cells was performed 24 h post transfection. Mock-transfected control was treated under identical conditions, with the exception of the absence of the mimic.

The MOs (Gene Tools, LLC) were designed and injected into fertilized one‐cell embryos, as detailed in Table S1. The specificity and inhibitory efficiency of each MO were determined as described previously (Eisen and Smith, 2008). Optimal MO concentrations (see Supporting Information Table S1) were determined on the basis of morphological criteria. miRIDIAN (Dharmacon) miRNA mimics for miR‐181 were injected at a final concentration of 2 μM. Embryos injected with mismatched MO‐miR‐181a/b or the negative mimic were used as controls.

Dual-luciferase reporter assay was performed according to Cristino, et al.22 (link). Briefly, a 3′UTR fragment containing the predicted MRE (or a control sequence with perfect match to miR-34) was anchored to the 3′end of Renilla reniformis luciferase coding gene in the PsiCHECK2 vector (Promega). Cos-7 cells were co-transfected with both vector and synthetic mimic ame-miR-34-5p (miRIDIAN, Dharmacon). After 24 hours, the luciferase activity was measured using Dual Luciferase Assay (Promega), according to manufacturer’s instructions. Firefly luciferase activity was used to normalize Renilla luciferase activity. All transfections were performed in six replicates each time. We used two samples t-test to determine the difference between two conditions (t-test, p < 0.05). The complete method procedure used in the Luciferase-reporter assays is described in Supplementary File S1; the primers used to amplify the 3′UTR fragments and the mimic miRNAs are listed at Supplementary Table S6.