Genomic dna screentape system

Leukocyte DNA was extracted from EDTA blood samples, using the DNA Isolation kit for Mammalian Blood (Roche). DNA quality was assessed on a Genomic DNA ScreenTape system (Agilent) and quantified using a Qubit 3.0 Fluorometer (Thermofisher). DNA was treated by bisulphite and then hybridized to the Infinium MethylationEPIC BeadChip (Illumina; ~850 000 sites), starting from 500 ng of DNA. All experiments were performed following the manufacturer’s instructions at the P3S Post-Genomic Platform of Sorbonne University (Paris, France).

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.

The pleural fluid was centrifuged at 3000 g for 15 min to remove cellular debris. The exosomes were isolated from the supernatants using ExoQuick™. A nucleic acid of exosomes was extracted with the MagMAX™ Total Nucleic Acid Isolation Kit. cDNA synthesis was performed using a SuperScript™ VILO™ cDNA Synthesis Kit. We extracted the total DNA from the cell pellet using the QIAamp DNA Mini Kit (QIAGEN, Hilden, Germany). The sizes of the DNA fragments in the exosomes were assessed using a 2200 TapeStation Instrument (Agilent Technologies, Santa Clara, CA, USA) with the Genomic DNA ScreenTape System. The DNA concentration was assessed with the Qubit 3.0 Fluorometer (Thermo Fisher Scientific). The RNA yield and size distribution were analyzed using an Agilent 2100 Bioanalyzer with an RNA 6000 Pico kit (Agilent Technologies, Foster City, CA, USA).

Leukocyte DNA was extracted from EDTA blood samples, using the DNA Isolation kit for Mammalian Blood (Roche, Basil, Switzerland). DNA quality was assessed on a Genomic DNA ScreenTape system (Agilent, Santa Clara, CA, US) and quantified using a Qubit 3.0 Fluorometer (Thermofisher, Waltham, MA, US). DNA was treated by bisulfite and then hybridized to the Infinium MethylationEPIC BeadChip (Illumina, San Diego, CA, US; ~ 865,000 sites), starting from 500 ng of DNA. All experiments were performed following the manufacturer’s instructions at the P3S Post-Genomic Platform of Sorbonne University (Paris, France).