Elution and analysis of RNA samples

Beads with hybrids were magnetically separated using a Dynal magnet and the supernatant was discarded. Beads were then resuspended by pipetting in 50 μl NLS RNA Elution Buffer (20 mM Tris pH 8.0, 10 mM EDTA, 2% NLS, 2.5 mM TCEP). To release the target RNA, beads were heated for 2 min at 95°C. Beads were then magnetically separated and the supernatants containing eluted target RNA were digested by the addition of 1 mg/ml proteinase K for 1 hr at 55°C to remove all proteins. The remaining nucleic acids were purified using the RNA Clean and Concentrator Kit (Zymo).

For synthesis of cDNA, the Maxima First Strand cDNA Synthesis Kit for RT-qPCR (Thermo Fisher Scientific) was used following manufacturer's protocol. To quantify RNA enrichment, RT-PCR was performed in triplicate using Luna Universal qPCR Master Mix (New England Biolabs) with variable amounts of cDNA and 0.5 μM of target-specific primers in a CFX connect instrument (Bio-Rad). Primer sequences are shown in Supplementary file 1.