MeRIP-seq

The MeRIP procedure was performed according to Dominissini et al. (2012) with slight modifications. Briefly, the mPFC was dissected from the mice 2 h after behavioral training. Total RNA with DNase-treatment was extracted using the Direct-zol RNA mini-prep kit (Zymo). Eighteen micrograms of RNA were fragmented to ∼100 nt using ZnCl₂, followed by ethanol precipitation. Before immunoprecipitation (IP), fragmented RNA was denatured at 75°C for 5 min, chilled on ice for 5 min, and then resuspended in 500 μl IP buffer containing the following (in mm): 150 NaCl, 10 Tris-HCl pH 7.4, 100 ribonuclease-vanadyl complex, 0.1% Igepal CA-630, and 200U RNAseOUT). All steps were performed at 4°C unless otherwise stated. Ten percent of the sample was saved as the input RNA fraction and the remainder was subjected to two rounds of IP, each with 3 μg of rabbit anti-m⁶A antibody pre-coupled to 30 μl of sheep anti-rabbit Dynabeads (Invitrogen) in IP buffer for 1 h. IP beads were combined and washed four times with IP buffer. Bound RNA was eluted in 300 μl elution buffer (5 mm Tris-HCl pH 7.5, 1 mm EDTA, 0.05% SDS) and 4.2 μl proteinase K (20 mg/ml), followed by Trizol (Invitrogen) extraction.

Libraries were generated using the Scriptseq v2 RNA-Seq kit (Epicenter) according to the manufacturer's ultra-low input protocol. Ribosomal RNA (rRNA) was depleted from input RNA with Ribo-Zero (Epicenter). Fifteen cycles of PCR were performed on the cDNA, each with unique index primers. Equal amounts of each library were pooled and subjected to AMPure XP (Beckman Coulter) purification to remove primer-dimers. The final libraries (size distribution from 150 to 400 bp, with the peak at ∼220 bp) were run on a single-flow cell on HiSeq 2000 (Illumina) for paired-end 100 bp sequencing. Standard Illumina Genome Analyzer software (Casava v1.8.2) and pipelines developed in-house were used for image processing and sequence extraction.