Zymo rna clean and concentrator 5

All mRNA generating plasmids were digested with PvuII-HF (NEB R[Δ4]151 L) and ApaLI-HF (NEB R0507L) at 37 °C for 3 h. After digestion, templates were run on a 1% agarose gel to confirm cutting and the reactions were purified with Zymo DNA Clean and Concentrator-5 (Zymo Research D4013). 1ug of linearized template was used in a 100 uL T7 RNA Polymerase (purified in house) reaction that was incubated at 37 °C for 3 h. After T7 reactions were complete, 15uL TurboDNAse (ThermoFisher Scientific AM2238) was added, and reactions were incubated at 37 °C for 15 min. The RNA was then purified using a Zymo RNA Clean and Concentrator-25 Kit (Zymo Research R1017). Eluted RNA was measured using the Qubit RNA HS Assay Kit (Thermo Fisher Scientific Q32852) then capped following the protocol for the Vaccinia Capping System (NEB M2080S) and purified one last time using Zymo RNA Clean and Concentrator-5 (Zymo Research R1013). Capped RNA concentrations were measured using the Qubit RNA HS Assay Kit (Thermo Fisher Scientific Q32852). Final RNA was diluted to 0.25pmoles/ul for use in the in vitro translation assays.
For the comparing capped versus uncapped mRNA, uncapped RNA was incubated for 5 min at 65 °C to match the treatment of capped RNAs. The same RNA that was used in the vaccinia capping reaction was directly compared to the post-cap RNA.

M6A RIP was performed using EpiMark® N6-methyladenosine Enrichment Kit (NEB) following the manufacturer’s instructions with some modifications. Purified RNA was sheared to <350 bp using a Covaris ME220 and then purified using Zymo RNA Clean and Concentrator 5 (Zymo) following the manufacturer’s instructions. Purified RNA was then measured for concentration using the Qubit BR RNA Assay (ThermoFisher Scientific) following the manufacturer’s instructions, and 1 µg was used for each pulldown with 2 µL of M6A antibody. Following 1 h incubation and washes, RNA was eluted and purified with MyOne Silane Beads (ThermoFisher Scientific) then prepared for reverse transcriptase in which 1 µL from pulldown or input was put in reverse transcriptase reaction. Resulting cDNA was then used for qPCR using primers designed to predict M6A sites located on BDNF-AS (Supplementary Table 2). M6A sites were predicted using SRAMP^{37 (link)}. The data were analyzed using the ∆∆Ct method, normalizing to input, and the data are expressed as fold change relative to controls.