Feature extraction software version 11.0.1.1

Total RNA was digested with RNase R (Epicentre, USA) to remove linear RNAs and enrich circRNAs. Then, the enriched circRNAs were amplified and transcribed to fluorescent cRNA utilizing a random priming method (Arraystar Super RNA Labeling Kit; Arraystar; USA). The labeled cRNAs were hybridized to an Arraystar Human circRNA Array (8x15K, Arraystar). After washing the slides, the arrays were scanned with an Agilent G2505C scanner. Agilent Feature Extraction software (version 11.0.1.1) was used to analyze the acquired array images. Quantile normalization and subsequent data processing were performed using the limma package in R software. CircRNAs with statistically significant differential expression between the two groups were identified through volcano plot filtering. Differentially expressed circRNAs between two samples were identified through fold change filtering. Hierarchical clustering was performed to visualize the distinguishable circRNA expression patterns among samples.

Total RNA was isolated from the cells of each group using TRIzol reagent (Invitrogen). RNA expression profiling was then performed using the Agilent mouse lncRNA + mRNA microarray V2.0 platform. The arrays were scanned by the Agilent G2565CA Microarray Scanner. Agilent Feature Extraction software (version 11.0.1.1) was used to analyze acquired array images. Quantile normalization and subsequent data processing were performed using the GeneSpring GX v11.5.1 software package (Agilent Technologies). Differentially expressed genes were identified by fold change filtering.

Immunoprecipitated RNA samples of the HD‐MSCs and AML‐MSCs were labelled with Cy5 fluorescent dye using Super RNA Labelling Kits (Arraystar Inc., Rockville, MD, USA) and then purified using RNeasy Mini Kits. The Cy5‐labelled cRNAs were fragmented and hybridised to a human mRNA and lncRNA m⁶A epitranscriptomic microarray (8 × 60 K; Arraystar) containing 44 122 mRNA and 12 496 lncRNA degenerate probes. The hybridised arrays were scanned using a G2505C Scanner (Agilent Technologies Inc., Santa Clara, CA, USA) [29]. All spots on the microarray were evaluated using Feature Extraction Software Version 11.0.1.1 (Agilent Technologies Inc.). The raw intensity of immunoprecipitated RNAs was normalised using an average of log₂‐scaled spike‐in RNA intensities. The fold changes between the HD‐MSCs/AML‐MSCs were determined for each transcript, and P‐values were calculated. Differentially m⁶A‐methylated RNAs were identified using a cut‐off of fivefold (P < 0.05). Differentially m⁶A‐methylated mRNA transcripts were identified using Gene Ontology, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses and Gene Set Enrichment Analysis (GSEA) [30].

Total RNA isolated from BGC-823 cells transfected with control and METTL3-overexpressing lentivirus was subjected to m6A-mRNA and lncRNA. Epitranscriptomic microarray and transcriptome sequencing were performed by Aksomics Inc. (Shanghai, China) and Beijing Genomics Institute (BGI, China), respectively. Agilent Feature Extraction software (version 11.0.1.1) analyzed the acquired array images. Raw intensities of IP (immunoprecipitated, Cy5-labeled) and Sup (supernatant, Cy3-labeled) were normalized with an average of log2-scaled spike-in RNA intensities. After spike-in normalization, the probe signals with Present (P) or Marginal (M) QC flags in a certain proportion were retained for m6A quantification based on the IP (Cy5-labeled) normalized intensities. Differentially m6A-methylated RNAs between two comparison groups were identified using the screening criteria for the fold change and statistical significance (p-value) thresholds. Hierarchical clustering was performed to show the differential m6A-methylation patterns among samples.

The Arraystar Human Circular RNA Microarray V2.0 (Arraystar, Inc.), performed by Kang Chen Biotech (Shanghai, China), was designed for the purpose of profiling circRNAs in the human genome. Scanned image processing was analysed using Agilent Feature Extraction Software Version 11.0.1.1. CircRNAs (|fold-change|≥ 2.0 and P-value < 0.05) were selected as markedly differentially expressed circRNAs. The microarray data produced in this study have been uploaded to the NCBI/GEO repository (Accession Number: GSE145610).

Total RNA was extracted from five paired CRC tissues and NCTs using TRIzol Reagent (Invitrogen, USA). RNA quantity and quality were measured by NanoDrop ND-1000. RNA integrity was assessed by standard denaturing agarose gel electrophoresis. LncRNA microarray was performed by (Aksomics, Shanghai, China). Arraystar Human LncRNA Microarray V3.0 is used for detecting the global profiling of human LncRNAs. Agilent Feature Extraction software (version 11.0.1.1) was used to analyze acquired array images. LncRNAs were deemed differentially expressed if their fold change between the CRC and NCT (normal colon tissue) groups exceed 2.0 and their P-values were less than 0.05.