miR-eCLIP of MCF10A cells following treatment with MIR-28 family microRNA mimics

To identify in vivo RNA targets of the MIR-28 family microRNAs hsa-miR-28-5p or hsa-miR-708-5p in MCF10a human breast epithelial cells, we profiled microRNA mimic:cellular RNA chimeras using miR-eCLIP. MCF10A cells were seeded at 3.5E6 cells/dish in two 15 cm dishes for 48 h, then transfected with microRNA mimics for 27 h. Each dish was treated with one MIR-28 family microRNA mimic, either hsa-miR-28-5p or hsa-miR-708-5p (Horizon Discovery/Dharmacon). Approximately 10E6 cells from each treatment were UV crosslinked and pooled together for AGO2 immunoprecipitation, RNA isolation, library preparation, and next-gen sequencing by Eclipse Bioinnovations (San Diego, CA). Single-end 122 bp sequencing was performed on the NextSeq platform.