Profiling circRNA Expression in Embryonic Mouse Brain

CircRNA expression in E18 whole brain was profiled using Arraystar Mouse Circular RNA Microarray service (Arraystar Inc., Rockville, MD). The circRNA array platform consisted of 14,236 probes designed by the manufacturer to detect the unique circRNA splice junction utilizing several circRNA sources (Memczak et al., 2013 (link); Guo et al., 2014 (link); You et al., 2015 (link)). Briefly, 800 ng of extracted total RNA (see above) was treated with RnaseR (3 h at 37°C of ribonuclease R, 20 U/μL, Epicenter, Madison WI) to digest linear RNA and enrich circRNA. Random primers were then used to amplify and transcribe the enriched circRNAs into fluorescent cRNAs per Arraystar Super RNA Labelling protocol (Arraystar Inc.). Using Arraystar Mouse Circular RNA arrays (8 × 15K, Arraystar, Inc.), the labeled cRNAs were hybridized and incubated in an Agilent hybridization oven for 17 h at 65°C (Agilent Technologies, Santa Clara, CA). The slides were washed and eventually scanned using the Agilent Scanner G2505C (Agilent Technologies). All circRNA profiling data have been deposited in the Mendeley online data repository¹.