MeRIP-seq and RNA-seq

The sequencing experiments were conducted at Cloud-seq Biotech Inc. (Shanghai, China). First, we collected and quantified total RNA from the sciatic nerve tissues of control (uninjured) and SNI (7 days after injury) groups. Then, Arraystar Seq-StarTMpoly(A) mRNA Isolation Kit (Arraystar, MD, United States) was used to extract poly(A) mRNA from total RNA. The tubes with 1 μg/μL RNA and fragmentation buffer (metal-ion) were incubated at 70°C for 6 min in a preheated thermal cycler block. Subsequently, 30 μL of 3 M sodium acetate (NaOAc), 1 μL of glycoBlue, and 750 μL volumes of 100% ethanol were added, mixed and kept at −80°C overnight. After centrifugation and pellet resuspension, RNA fragmentation was conducted by running 0.5 μg of RNA through 1.5% (wt/v) agarose gel. The fragmented RNA was immunoprecipitated with magnetic beads containing m6A antibody (1 μL m6A antibody in 50 μL 1 × IP buffer) using the GenSeq^TM m6A RNA IP kit (GenSeq Inc., China). The input samples without immunoprecipitation were used as a control group. The following reagents were used for elution and purification in sequence: 1 × IP buffer, LB Buffer, HS Buffer, RLT Buffer, 100% and 75% ethanol.

MeRIP-seq libraries were constructed using the NEBNext^® Ultra II Directional RNA Library Prep Kit (New England Biolabs, Inc., United States). For RNA-seq, total RNA (1 μg) was used to remove the rRNAs via Ribo-Zero rRNA Removal Kit (Illumina, San Diego, CA, United States). RNA-seq libraries were constructed using the TruSeq Stranded Total RNA Library Prep Kit (Illumina, San Diego, CA, United States). Then, both libraries were controlled and evaluated with the Bio Analyzer 2100 system (Agilent Technologies, United States). The 10 pM libraries were denatured as single-stranded DNA molecules and amplified in situ as clusters. MeRIP-seq and RNA-seq procedures were carried out on an Illumina NovaSeq 6000 instrument with 150-bp paired-end reads. The quality control metrics of the sequencing data are listed in Supplementary Table 1.