Nd 1000

Total RNA from OS sample and case matched normal tissues was isolated using TRIzol (Invitrogen, MD, USA) and quantified using the NanoDrop ND-1000. The sample preparation and microarray hybridization were performed based on standard Arraystar’s protocols (Aksomics, Shanghai, China). Briefly, total RNA was digested with RNase R (Epicentre, Inc.) to remove linear RNAs and enrich circular RNAs. Then, the enriched circular RNAs were amplified and transcribed into fluorescent cRNA utilizing a random priming method (Arraystar Super RNA Labeling Kit; Arraystar). The labeled cRNAs were hybridized onto the Arraystar Human circRNA Array V2 (Arraystar). After washing the slides, the arrays were scanned by the Agilent Scanner G2505C.

Total RNA was first isolated and purified with TRIzol (Invitrogen, America), and then, the amount and purity of total RNA were measured with NanoDrop ND-1000. The integrity of the RNA was tested by Bioanalyzer 2100, and the verification was conducted through agarose electrophoresis. Validation criteria were shown as follows: concentration > 50 ng/μl, RIN > 7.0, OD260/280 > 1.8, and total RNA > 1 μg. After reverse transcription of total RNA into cDNA, E. coli DNA polymerase I (New England Biolabs, America) was applied for double-strand synthesis. Specifically, the complex duplex of DNA and RNA was converted into a DNA duplex, and at the same time, dUTP solution was incorporated into the duplex to supplement the ends of the duplex. And the A nucleobases were added at the ends of the double-stranded DNA to link with T nucleobases. Then, the fragment sizes were screened and purified with magnetic beads. After the duplex was digested by Uracil-DNA Glycocasylase (UDG) (New England Biolabs, America), libraries with a fragment size of 300 bp ± 50 bp were formed through PCR. Finally, with PE150 as the sequencing mode, double-end sequencing was performed according to the instructions of the Illumina NovaSeq™ 6000 kit.

LGs were dissected out as previously described (Finley et al., 2014 (link)). Total RNA was extracted from whole LGs (mesenchyme and epithelium) of embryonic (E18) and adult (34 weeks of age) animals. E18 samples were composed of at least 10 LGs (5 individuals) pooled together. Biological triplicates for each sample were included in the analysis. The quality and concentration of the extracted RNA was monitored using a nanodrop spectrophotometer (ND-1000, Fisher Scientific). The transcriptomic analysis was performed by the Functional Genomics Unit (FuGU, Helsinki, Finland). Samples were hybridized on Affymetrix GeneChip® Mouse Transcriptome Assay 1.0 (Affymetrix, Santa Clara, CA, USA) microarrays, and data analysis was performed with the Affymetrix Transcriptome Analysis Console (TAC) Software.

RNA was extracted from ∼100 mg of ground tissue using 1 ml of TRIzol (TRI Reagent, Sigma) according to the manufacturer's instructions. RNA concentration was measured using a nanodrop spectrophotometer (ND-1000, Fisher Scientific). RNA quality was visualized by gel electrophoresis on 1% agarose and ethidium bromide gel staining. Small RNA from immunopurified AGO1 was extracted by TRIzol as above, but was precipitated from the aqueous phase after chloroform extraction in the presence of 10 μg of glycogen as a carrier.

RNA extraction from cells was performed using the EZ-RNA II Isolation Kit (Biological Industries Beit Haemek, Israel) according to the manufacturer's instructions. Extracted RNA was quantified by the NanoDrop spectrophotometer with ND-1000 software (Thermo scientific, MA, USA). The RNA extraction from exosomes was executed using the Total Exosome RNA and Protein Isolation Kit (Invitrogen, CA, USA) according to manufacturer's instructions.