GSM9321770: MEC1 cells, Biotinylated miR-scramble mimics, 48h, rep4; Mus musculus; RNA-Seq
Transfected epicardial cellswere harvested 48 h post-transfection and lysed using the miRNeasy Tissue/Cells Advanced kit lysis buffer. Complexes of biotinylated RISC and associated mRNAs were captured using streptavidin-coated magnetic beads with background reduction achieved via Argonaute immunoprecipitation A total of 2.5 ng of RNA was used to generate cDNA using the SMART-Seq v4 Ultra Low Input RNA Kit (Clontech-Takara). From this, 1 ng of amplified cDNA was used to prepare barcoded sequencing libraries with the Nextera XT DNA Library Preparation Kit (Illumina), in which cDNA fragmentation and adapter ligation occur in a single reaction, followed by amplification and cleanup. Library size was assessed using the Agilent 2100 Bioanalyzer High Sensitivity DNA chip, and concentrations were determined with a Qubit® fluorometer (Thermo Fisher Scientific).