m6A RNA Immunoprecipitation and qRT-PCR Analysis

The relative abundance
of occludin (OCLN), claudin 1 (CLDN1), zonula occluden (ZO-1), and
heme oxygenase-1 (HO-1) mRNA in m⁶A antibody IP samples
and input samples was assessed by qRT-PCR. Total RNA was isolated
from mucosa with TRIzol reagent (Vazyme) followed by polyadenylated
RNA extraction using Dynabeads mRNA DIRECT (Invitrogen) kit. A 200
ng aliquot of mRNA was saved as input samples. The remaining mRNA
was used for m⁶A-immunoprecipitation as given in Dominissini
et al.^{14 (link)} and Zhong et
al.^{26 (link)} A 5 μg aliquot of mRNA was
incubated with m⁶A antibody (Abcam) in IP buffer (10 mM
Tris HCl, pH 7.4, 150 mM NaCl, and 0.1% Igepal CA-630) supplemented
with RNase inhibitor (Fermentas) for 2 h at 4 °C. Dynabeads Protein
A (Invitrogen) was added to the solution and rotated for an additional
2 h at 4 °C. After washing with IP buffer, mRNA was eluted from
the beads via incubation in 300 mL elution buffer (0.1 M NaCI, 10
mM Tris, pH 8.0, 1 mM ethylenediaminetetraacetic acid, 0.05% sodium
dodecyl sulfate (SDS), 200 mg/mL proteinase K) for 1.5 h at 50 °C.
Finally, m⁶A IP mRNA was recovered by ethanol precipitation,
purified by phenol/chloroform extraction, and analyzed by qRT-PCR.
Primer sequences are listed in Table S3.