Picogreen

DNA was extracted from frozen soil samples using the PowerSoil DNA Isolation Kit (MO BIO Laboratories, Inc., Carlsbad, CA, USA) according to the recommended protocol for highly organic soil. Approximately 0.15 g of soil from each sample was used for isolation of DNA. Quantification was performed with the standard dsDNA quantification protocol for PicoGreen (Thermo Fisher Scientific, Inc., Waltham, MA, USA). All pipetting for DNA extraction was conducted with an Eppendorf epMotion 5075 pipetting robot (Eppendorf AG, Hamburg, Germany). We amplified 16 S ribosomal RNA (rRNA) gene sequences in duplicate from the extracted DNA. The PCR primers used are those described in Caporaso et al. (2012) that target the bacterial/archaeal 16S rRNA gene variable region 4 (515 F/806 R) for downstream paired-end Illumina (Illumina, Inc., San Diego, CA, USA) barcoded sequencing [15 (link)]. Amplicons were quantified with PicoGreen, and 200 ng of each sample was pooled and purified with the desalting protocol of the Qiagen QIAquick Spin Filter Purification Kit (Qiagen Inc., Valencia, CA, USA). The amplicon pool was submitted to the Cornell Life Sciences Sequencing Core with the sequencing primers detailed in Caporaso et al. (2012).

For DNase treatment, 20 μL of cell free serum was incubated with 10 mU of recombinant DNase (Qiagen) for 30 min at 37 °C followed by detection with PicoGreen™ reagent. EVs from serum were isolated using the Total Exosome Isolation Kit from serum (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s recommendations.

DNA was quantified using Picogreen (Invitrogen by Thermo Fisher Scientific) and 1 μg of genomic DNA was bisulfite-converted using EZ DNA Methylation according to manufacturer’s protocol (Zymo Research, Irvine, CA, USA). Briefly, DNA was bisulfite-converted for 16 h at 50°C and subsequently desulfonated, washed, and eluted in 10 μl of elution buffer. The bisulfite converted region of interest was amplified by the high-fidelity DNA polymerase KOD -Multi & EPi (Toyobo, Osaka, Japan) using specific primers (Supplementary Table 1). PCR thermal profile was: 95°C for 4 min; 35 cycles of 95°C for 30 s, 55°C for 30 s, 72°C for 30 s; 72°C for 7 min. PCR products were purified by QIAquick PCR columns (Qiagen, Hilden, Germany), quantified using Picogreen and confirmed through agarose gel electrophoresis.