Miridian mimic

Antisense oligonucleotides were designed to target sdRNAs and ordered as custom IDT^® miRNA Inhibitors from IDT (Integrated DNA Technologies, Coralville, IA, USA). Similarly, sdRNA mimics and scrambled controls were ordered as custom miRIDIAN mimics from Dharmacon (GE Healthcare Dharmacon, Inc., Chicago, IL, USA). Mimic and inhibitor sequences are detailed in Supplementary File S2. Cell migration, proliferation, and invasion assays were then performed to observe the effects of manipulating sdRNA-D19b and -A24 levels. Human PC3 cells (ATCC, CR L-1435) were cultured at 37 °C in 25 cm² vented flasks (Corning, Manassas, VA, USA) with DMEM (Corning) supplemented with 10% fetal bovine serum (Corning) and 1% PenStrep (Corning) in a humidified atmosphere at 5% CO₂. For transient transfections, the cells were cultured in 12-well plates and grown to 60% confluency before transfection with mimics or inhibitors using Lipofectamine RNAiMAX (Life Technologies, Carlsbad, CA, USA).

Cell proliferation assays were performed using the xCELLigence System (Roche Applied Science, Indianapolis, IN, USA), which allows the real time monitoring of cell proliferation. Briefly, 10,000 cells of JEG3 or HT-29 were transfected in the E-plate 16 wells (Roche Applied Science, Indianapolis, IN, USA) to a final concentration of 50 nM of miRIDIAN mimics (Dharmacon, Lafayette, CO, USA) in 200 μl of MEM containing 10% FBS. Culture medium was replaced every 24 hours. Cell index value was acquired at 24, 48, 72 and 96 h. All experiment conditions were done in triplicates.

The generation of MCF7-Ctrl and Six1 (ref. 62 (link)) lines, MCF12A-Ctrl and Six1 (ref. 21 (link)), and 66Cl4 Six1 KD lines15 (link) was previously described. For clonal isolates of MCF7 and MCF12A cell lines, each experiment shown displays one Six1 and one Ctrl clone due to space constraints, but each experiment is representative of data seen across at least two sets of clones. Transient gene KD experiments were performed using ON-TARGETplus SMARTpool siRNA constructs (Supplementary Table 2) against the gene of interest. Transient overexpression of Six1 was performed using a pcDNA3.1 vector containing full-length Six1 (ref. 47 (link)). eGFP-RPL26 and pGL3ctrl luciferase vector flanked by TP53 UTRs were gifts from Dr Kastan (Addgene plasmid #31980 and #28175). Mutations were introduced by site-directed mutagenesis with the use of the Quickchange II kit (Agilent Technologies). Transient overexpression of miRNAs was performed using miRIDIAN mimics (Dharmacon). All cell lines were profiled to confirm their identity and periodically checked for mycoplasma contamination. For cell culture conditions and drug additions, see Supplementary Tables 3 and 4.