The mRNA m6A methylation modifications in lung tissues from smoke-exposed mice (n=3) and room air-exposed mice (n=3)

The Arraystar Mouse m6A-mRNA&lncRNA Epitranscriptomic Microarray (Arraystar Inc, Rockville, MD, USA) was utilized for the analysis of mRNA m6A methylation modifications in lung tissues from smoke-exposed mice (n=3) and room air-exposed mice (n=3). Total RNA from these tissues was first immunoprecipitated using an anti-N6-methyladenosine (m6A) antibody for the separation of RNA into two groups: 'IP', comprising m6A-modified RNAs isolated from magnetic beads, and 'Sup', consisting of unmodified RNAs obtained from the supernatant. These RNA fractions were then labeled with Cy5 (for IP) and Cy3 (for Sup) in separate reactions according to the Arraystar RNA Labeling protocol, producing complementary RNAs (cRNAs). These labeled cRNAs were subsequently combined and hybridized onto the Arraystar Mouse mRNA&lncRNA Epitranscriptomic Microarray. Following hybridization, the microarray slides were washed and scanned using an Agilent Scanner G2505C in two-color channels.The acquired array images were analyzed using Agilent Feature Extraction software (version 11.0.1.1; Santa Clara, CA, USA). The raw intensity data of both IP (Cy5-labeled) and Sup (Cy3-labeled) samples were normalized against the average of log2-scaled Spike-in RNA intensities. After this normalization, probes with 'Present' (P) or 'Marginal' (M) QC flags in at least three out of the six samples were selected for further analysis of 'm6A quantity', which was determined based on the normalized intensities of the IP samples. The mRNA expression level was calculated for the IP (Cy5-labelled) and Sup (Cy3-labelled) normalized intensities. The identification of differential mRNA levels and m6A-methylated RNAs between the two groups was conducted by applying thresholds for fold change and statistical significance (p-value). Hierarchical Clustering was then performed to illustrate the distinct patterns of m6A methylation among the samples.