Streptavidin mag sepharose beads

RNA antisense purification was performed following a protocol from the Guttman lab.^[79] In brief, probes were generated by in vitro transcribing biotinylated antisense ARGI followed by controlled RNA fragmentation using a RNA Fragmentation Reagent (Invitrogen). Probes against a non‐related lncRNA of similar size were used as control. Extracts from EndoC‐βH1 cells were crosslinked adding 2 mm disuccinimidyl glutarate for 45 min at room temperature and subsequently 3% formaldehyde for 10 min at 37 °C. Crosslinked RNA‐chromatin was fragmented by sonication, hybridized with control or ARGI‐specific RNA probes, captured using Streptavidin Mag Sepharose beads (Cytiva), washed extensively, and eluted. Retrieved RNA was purified using PureLink Micro RNA columns (Thermo Scientific) and fragmented DNA by NucleoSpin Gel and PCR Clean‐up (Macherey‐Nagel). Enrichment of ARGI in the regulatory regions of IFNβ and ISG15 was quantified by qPCR.

SMART-MSP (Sensitive Melting Analysis after Real-time Methylation-Specific PCR) reactions were performed in technical duplicates on a Mic qPCR Cycler (BMS, Sydney, Australia) as previously described [51 (link)] to specifically amplify only methylated epialleles. The primer sequences (Bioneer) can be found in Additional file 1: Table S1. The amplified methylated epialleles were then pyrosequenced on a Qseq instrument (BMS) using the Q48 Advanced CpG kit (Qiagen) and Streptavidin Mag Sepharose beads (Cytiva, MA, USA) to assess the genotypes of the SNPs on only the methylated epialleles. The pyrosequencing data were analysed with Qseq software 2.4.4 (BMS).