Rna clean and concentrator 5 spin column

Total RNA of L4/L5 DRGs was isolated from WT and
Syn1-Cre;Mettl14^f/f cKO mice, extracted
with Trizol reagent (ThermoFisher; 15596018) and recovered by RNA Clean and
Concentrator-5 spin columns (Zymo; R1015). The RNAseq library was prepared
using the Smart-seq2 protocol. The distribution of fragment sizes was
verified. Libraries of three biological replicates under different
conditions were uniquely barcoded, pooled at equimolar concentrations, and
sequenced on Illumina NextSeq 500 (Su et
al., 2017).

mRNA from total RNA of adult mouse DRGs was purified with Dynabeads
Oligo (dT)₂₅ (ThermoFisher; 61006). Five μg of
anti-m⁶A polyclonal antibody (Synaptic Systems; 202003) was
conjugated to Dynabeads Protein A (ThermoFisher; 10001D) overnight at
4°C. A total of 150 ng of mRNA was then incubated with the
antibody/beads in 1× IP buffer (10 mM Tris-HCL, 150 mM NaCl, and
0.1% (vol/vol) Igepal CA-630), supplemented with 200 U SUPERase In
RNase Inhibitor (ThermoFisher; AM2696), for 2 hr at 4°C. After incu
bation, the beads were washed 3 times with IP buffer and the m⁶A
RNA was eluted twice with 6.7 mM
N⁶-Methyladenosine (Sigma-Aldrich; M2780) in
1× IP buffer. The eluted RNA was extracted with Trizol reagent
(ThermoFisher; 15596018) and recovered by RNA Clean and Concentrator-5 spin
columns (Zymo; R1015). The equivalent amount of input and
m⁶A-IPed RNA were prepared for library generation using the
SMART-seq protocol as described (Picelli et
al., 2014). Two biological replicates of naïve and SNL
conditions were sequenced using Illumina NextSeq 500.

After labeling, total cellular RNA was harvested using Trizol Reagent (Invitrogen) following the manufacturer’s instructions or RIPA lysis buffer (150 mM NaCl, 5 mM EDTA pH 8.0, 50 mM Tris, pH 8.0, 1.0 % NP-40, 0.5 % sodium deoxycholate, 0.1% SDS) spiked with DNase Turbo and RNaseOut (Thermo Fisher). Briefly, ~20 million cells were scraped and suspended in 2 mL of lysis buffer, and incubated on ice for 15 min. Lysates were mixed 1:1 with phenol/chloroform pH 5.2 (Fisher) vortexed briefly and spun down at 4 °C to separate the layers. The aqueous layer was then applied to a Zymo RNA clean and concentrator IIC column (Zymo) and total RNA was isolated according to the manufacturer’s instructions, with RNA eluted in 50 μL of nuclease free water. IEDDA reactions were prepared using 10 μg of total RNA in solutions of 6% DMSO:AcOH (pH 5.0) water, and 1 mM Tz-4 biotin to a final volume of 50 μL. Reactions were incubated with at 37 °C for 2 h at 400 RPM. Biotinylation reactions were purified using Zymo RNA clean and concentrator-5 spin column according to the manufacturer instructions, with RNA eluted in 10 μL of nuclease free water.