Identification of RNAs bound in cellulo to miR-491-5p

We purified all RNAs in direct interaction with biotinylated versions of miR-491-5p or a control miRNA in the cytoplasmic compartment (see protocol) of IGROV1-R10 chemoresistant human ovarian cancer cell line. Input and purified RNA were deep-sequenced in order to calculate an enrichment score able to identify which transcripts were bound in cellulo by miR-491-5p transcript enrichment was calculated as follow after normalization: (biotin-miR-491-5p Pull-Down/biotin-cel-miR-65 Pull-Down) / (biotin miR 491-5p Input/biotin-cel-miR-Input). This method of enrichment calculation is aimed to normalize for the decrease in miR-491-5p-targeted RNAs levels in biotin-miR-491-5p transfected samples relative to biotin-cel-miR-65. The Pull-Down experiment was run 3 times independently. The RNA from the 3 experiments was pulled together to constitute the samples submitted to NGS.