Scanner g2505c

TRI Reagent (catalogue no. T9424; Sigma, Germany), miRNeasy Micro Kit (cat alogue no. T9424 217084; Qiagen, Germany), RNase-Free DNase Set (cat alogue no. 79254; Qiagen), Tiangen miRcute miRNA cDNA First-Strand Synthesis Kit (cat alogue no. KR201; Tiangen, Beijing, China), SYBR Green PCR kit (cat alogue no. Fp411-02; Tiangen, Beijing, China), Agilent Bioanalyzer 2100 analyzer (Agilent Technologies, Santa Clara, CA, USA); Scanner G2505C (Agilent Technologies), feature extraction (version 10.7.1.1, Agilent Technologies), data processing software Genespring (version 13.1, Agilent Technologies), and Affymetrix Agilent Human miRNA, Release 21.0 (8 × 60K, Design ID: 070156) chip were utilized for this study.

For microarray analysis, we used the Agilent Array platform version 5.7 to amplify and transcribe the total RNA from each sample into fluorescent cRNA. The labelled cRNAs were hybridized using the Rat LncRNA 4 × 44K Array v2.0. We scanned the arrays using the Agilent Scanner G2505C and used the GeneSpring GX v11.5.1 software package and Agilent Feature Extraction software (version 11.0.1.1) to analyse the acquired array images and perform data processing.

Total RNA from each sample was amplified and transcribed into fluorescent cRNA according to Agilent Technologies' Quick Amp Labeling protocol (version 5.7). The labeled cRNAs were hybridized onto a Whole Human Genome Oligo Microarray (4×44K; Agilent Technologies). After washing the slides, the arrays were scanned using an Agilent Technologies Scanner G2505C. Agilent Technologies 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 GeneSpring GX v11.5.1 software package (Agilent Technologies). Differentially expressed genes were identified through fold change filtering. The selection criterion was > 1.5-fold difference in expression (upregulated expression > 1.5-fold; downregulated expression < 0.67-fold). GO analysis (Supplementary Methods) was used to determine the roles played by the differentially expressed genes in these biological GO terms.

CircRNAs were enriched by removing linear RNAs with Rnase R (Epicentre, Madison, WI, USA). Arraystar Human circRNA Array and LncPath Human Cancer Array were used for hybridization, and then the Agilent Scanner G2505C (Jamul, CA, USA) was used for scanning. Heat maps were developed using HemI1.0.1 software (http://hemi.biocuckoo.org/down.php). And then, the downstream signaling pathways of circ-METRN were also analyzed.

Total RNA was extracted from METTL3 knockdown SAS cells and the corresponding control cells, and was incubated with an anti-m6A antibody and magnetic beads for immunoprecipitation. After eluting, we tagged m6A modified RNA as “IP” and labeled with Cy5, and unmodified RNA in the supernatant as “Sup” and labeled as Cy3, they were then used as cRNAs in analysis using Arraystar Super RNA Labeling Kit. After hybridizing these cRNAs onto a Arraystar Epitranscriptomic Microarray slide (8 × 60 K, Arraystar) and washing it, an Agilent Scanner G2505C was used to scan the array. A normalization was done on the raw intensities of Cy5-labeled “IP” and Cy3-labeled “Sup” using an average of log2-scaled Spike-in RNA intensities. These intensities gave the basis to calculate m6A methylation level and quantity. Differentially m6A-methylated RNAs were filtered by fold change > 1.2 and p-value < 0.05, and then be used for hierarchical clustering, GO analysis and pathway analysis.

Total RNA of ALDH⁺ cells and ALDH^- cells derived from AMOC2 cells were isolated from collected cells using an RNeasy Mini Kit (QIAGEN, Valencia, CA) following the manufacturer's protocol. We used the commercially available Low Input Quick Amp Labeling Kit (Agilent Technologies). Purified total RNA (3 μg) was reverse-transcribed to generate double-stranded cDNA using an oligo dT T7 promoter primer and reverse transcriptase. Then cRNA was synthesized using T7 RNA polymerase, which simultaneously incorporated Cy3- or Cy5-labeled cytidine triphosphate. During this process, the samples of RNA derived from ALDH⁺ cells were labeled with Cy5, whereas the RNA derived from ALDH^- cells were labeled with Cy3 as control cells. Quality of the cRNA was checked using the Nano Drop. Cy3-labeled cRNA derived from ALDH⁺ cells and Cy5-labeled cRNA derived from ALDH^- cells were combined and then fragmented using a gene expression hybridization kit (Agilent Technologies). The cRNAs were hybridized to a 60-mer probe oligonucleotide microarray (G4845A human GE 4 × 44 k V2 Microarray kit) and incubated for 20 hours at 50°C. The fluorescent intensities were determined by an Agilent Technologies Scanner G2505C. Samples of ALDH⁺ cells were labeled with Cy3, whereas ALDH^- cells were labeled with Cy5. Microarray raw data and processed data have been deposited in the NCBI GEO database (GSE64539).

After extraction and purification, the total RNAs of the induced and noninduced groups (three cell samples per group) were analyzed by using microarray. Arraystar Human circRNA Array (Arraystar, USA) was used to perform RNA expression profiling. After RNAs were digested with RNase R, a random priming method (Arraystar Super RNA Labeling Kit; Arraystar) was used for the amplification and transcription of circRNAs to transfer into fluorescent cRNA. The arrays were analyzed by the Agilent Scanner G2505C, and Agilent Feature Extraction software (version 11.0.1.1) was used to access obtained array images.