Super rna labelling protocol

Profiling of circRNA expression in 100 human OFC postmortem brain (34 SCZ, 32 BD, and 34 Controls) was performed with the Arraystar Human Circular RNA Microarray (Arraystar Inc., Rockville, MD) per the manufacturer’s instructions with 13,617 probes designed to detect the unique circRNA splice junction based on numerous RNA-sequencing circRNA data [16 (link), 19 (link), 22 (link), 23 (link), 50 (link), 51 (link)]. Briefly 800 ng of total RNA previously quantified and quality verified (see above) were treated with an aggressive RnaseR treatment (3 h at 37 °C of ribonuclease R, 20 U/μL, Epicentre, Madison WI) to digest linear RNAs and enrich for circRNA expression. The enriched for circRNAs RNA was then amplified and transcribed into fluorescent cRNA via random primers according to the Arraystar Super RNA Labelling protocol (Arraystar Inc.). The labeled circRNAs were then hybridized onto the Arraystar Human Circular RNA arrays (8 × 15 K, Arraystar, Inc.) and incubated for 17 h at 65 °C in an Agilent hybridization oven (Agilent Technologies, Santa Clara, CA). Slides were then washed and scanned with the Agilent Scanner G2505C (Agilent Technologies). Differentially altered circRNAs as shown in Supplementary Tables 2–3. All circRNA profiling data have been deposited in the Mendeley online data repository: https://data.mendeley.com/datasets/9zdhc6pmx5/1.

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.