N6-Methyladenosine Enrichment Protocol

The EpiMark N6-Methyladenosine Enrichment Kit (item no. E1610S; New England Biolabs, Ipswich, MA) was used to enrich m⁶A modified RNA per manufacturer’s instructions, with minor modifications. RNA samples were fragmented at 94 °C for 3 min (Barros-Silva et al. 2020 (link)). Dynabeads™ Protein G magnetic beads from Thermo Fisher Scientific (product no. 10003 D) were washed and resuspended in Reaction Buffer. An anti-N6-Methyladenosine (m⁶A) rabbit monoclonal antibody (product no. 56593, Cell Signaling Technology, Danvers, MA) was bound to the beads, followed by the addition of the fragmented total RNA (3.7 μg) and RNasin ® Plus RNase Inhibitor (product no. N2611; Promega, Madison, WI). The normal Rabbit IgG control (product no. 2729, Cell Signaling Technology), an unconjugated rabbit polyclonal antibody, was used as a nonspecific IgG control for the immunoprecipitation. After the immunoprecipitation, RNA was eluted with Buffer RLT (product no. 79216; Qiagen) then cleaned and concentrated using Dynabeads™ MyOne™ Silane (product no. 37002 D; Life Technologies, Carlsbad, CA). The RNA samples from both input and immunoprecipitated samples were examined by RT-qPCR to quantify m⁶A enrichment.