Agilent 2200 tapestation platform

Untreated LR-PCR amplicons were used as negative controls for methylation. To generate positive controls, the same amplicons were treated in vitro with the recombinant CpG methyltransferase M.SssI (NEB). Briefly, 1 μg of amplicon DNA per 50μl reaction was treated for 4 h at 37°C with 50 units of M.SssI in the presence of 1× NEB buffer #2 and 160μM of S-adenosylmethionine (SAM). To test the efficiency of the M.SssI reaction, 10 units of methylation-sensitive restriction enzyme BstUI were added at the end of the incubation. This was followed by a further incubation at 60°C for 1 h. Protection of the M.SssI-treated amplicons from BstUI digestion was assessed using the Genomic DNA ScreenTape System (Agilent) on an Agilent 2200 TapeStation platform following manufacturer's instructions (data not shown). Supplementary Figure S1. To generate positive controls with intermediate methylation levels, we mixed negative and positive controls according to Supplementary Table S2.

Total RNA (10 ng) from serum-derived EVs was extracted and in turn inspected by Qubit2.0 software (Life Technologies, USA) on the Agilent 2200 TapeStation platform (Agilent Technologies, Santa Clara, CA, USA) for sample quality control. Agilent 2100 bioanalyzer electrophoresis system (Agilent Technologies, Santa Clara, CA, USA) was used for quantification of total RNAs. After that, library was constructed and the sequencing was carried out on HiSeq^TM 2500 platform according to the user guide using single end (1 × 50) standard sequencing program. The raw data was checked by C++ and R language as a quality control to obtain clean high-quality data. The expression of microRNA was analyzed by Perl software and the differential expression of microRNA was obtained by edgeR software. All the tests and heat map drawing were performed by Ribobio Comp (www.ribobio.com, Guangzhou, Guangdong, China). The raw data of microRNA deep sequencing assay in this study is available in the GEO (Gene Expression Omnibus) database with accession number GSE134205. Quality control results from deep sequencing including numbers of raw reads and mapping reads obtained were listed in Table S1.