Cpg methyltransferase

To evaluate if mt-cfDNA was recognized through TLR9, J774.1 cells were pre-treated for 4 h with 2.5 µM of TLR9 inhibitor (ODN 2088) or the control (ODN 2088 control, Thermo Fisher Scientific), for the quantification of pro-inflammatory cytokines.
To assess if unmethylated CpG motif of mtDNA was essential in promoting pro-inflammatory response, we prepared both totally unmethylated mtDNA and fully methylated mtDNA. We designed 8 primer pairs covering whole mtDNA and obtained totally unmethylated mtDNA fragments using conventional PCR with Ex Taq DNA polymerase (TaKaRa). The mtDNA fragments were incubated with M.SssI, CpG Methyltransferase (New England Biolabs, Beverly, MA, USA), and S-adenosyl methionine at 37 °C for 1 h. Before the application to J774.1, both unmethylated and methylated mtDNA were fragmented by using an ultrasonicator, as mentioned earlier to simulate cf-mtDNA.

The pCpGL vector containing a 0.53-kb fragment (−373 to +182) of the Aldh1a2 promoter and the double-stranded oligonucleotides for DNAP assay were methylated with CpG methyltransferase (M.SssI) and S-adenosylmethionine (New England Biolabs), according to the manufacturer's instructions. Mock methylation reactions did not contain M.SssI. Methylated and mock methylated vectors and oligonucleotides were purified by phenol-chloroform extraction and ethanol precipitation [25] (link).

Methylation of Avp promoter constructs was performed using CpG methyltransferase (M0226L; NEB) in accordance with the manufacturer's instructions. Mock methylation reactions were performed for all plasmids with identical reaction chemistry but with the omission of CpG methyltransferase. The methylation reactions were incubated at 37 °C for 8 h followed by 65 °C for 20 min. Plasmid DNA was purified using a QIAprep Spin Miniprep kit. To confirm methylation, plasmids were cut with methylation sensitive restriction enzyme PmlI (CACGTG), which recognises CpG site 5 within the Avp promoter of the pGL3‐Avp promoter constructs.

PCR-amplified Casp8AP2 promoters (4 μg) were incubated with 20 units of CpG methyltransferase (New England BioLabs) at 37 °C for 4 h in the presence of 160 μM SAM to induce methylation.

1 M Tris-HCl (pH8, molecular biology grade ultrapure, ThermoScientific, cat#J22638-K2), 2-deoxyadenosine (A) (>99%, FisherScientific, cat#AAJ6388606), Benzonase (Millipore Sigma, cat#E1014), CpG methyltransferase M.SssI(NEB, cat#M0226), dNTP set (Neta Scientific, cat#GHC-28-4065-51), HMW DNA Extraction kit (Promega, Cat#A2920), M. SssI (NEB, cat#M0226S), N6-methyl-2-dATP (TriLink, cat#N-2025), N6-methyl-2-deoxyadenine (m6A) (>99%, FisherScientific, cat#AAJ64961MD), Nanosep (MWCO 3 kDa, Pall, cat#OD003C33), phosphodiesterase I (Worthington, cat#LS003926), Quick CIP (NEB, cat#M0525S), Ultrapure distilled water (Invitrogen, cat#10977015). Taq methyltransferase (NEB, cat#M0219S), Dam methyltransferase (NEB, cat#M0222S), EcoRI methyltransferase (NEB, M0211S), ProNex^® Size-Selective Chemistry (Promega, cat#NG2001), PacBio Elution buffer (PacBio #101-633-500), ZORBAX Eclipse Plus C18 (Agilent, cat#959757-902). All reagents used for mass spectrometer analysis are molecular grade level or above. g-TUBEs, SMRTbell^® prep kit 3.0, REPLI-g Mini kit (Cat# 150023).

When indicated, dsDNA templates (2.0 μM) were treated with CpG methyltransferase (M.SssI, New England Biolabs) as instructed by the manufacturer. After the reaction, methylation was confirmed by resistance to restriction digestion by PvuI and SalI on template A and D, respectively (Supplementary Figure S6).

Genomic DNA (500 ng) extracted from melanoma cell lines, using QIAmp DNA Blood mini Kit (Qiagen, Hilden, Germany), was subjected to modification with sodium bisulfite using the EZ DNA Methylation-Gold Kit (Zymo Research, CA, United States). Primers for the analysis of the methylation status of LINE-1 were designed using the free on-line software MethPrimer (Li and Dahiya, 2002 (link)), and are the follows: LINE-1 Unmethylated F: 5′-TGTGTGTGAGTTGAAGTAGGGT-3′, Unmethylated R: 5′-ACCCAATTTTCCAAATACAACCATCA-3′; LINE-1 Methylated F: 5′-CGCGAGTCGAAGTAGGGC-3′, Methylated R: 5′-ACCCGATTTTCCAAATACGACCG-3′. SYBR green qMSP reactions were performed with methylated- or unmethylated-specific primer pairs on 2 μl of bisulfite-modified genomic DNA. The copy number of methylated or unmethylated sequences for LINE-1 gene was established by extrapolation from the standard curves. The percentage (%) of methylation was defined as ratio between methylated molecules and the sum of methylated and unmethylated molecules and data were reported as % of LINE-1 demethylation ± standard deviation (SD) of treated vs. untreated cells. CpG Methyltransferase (New England BioLabs, Ipswich, MA, United States) and RepliG mini Kit (Qiagen, Hilden, Germany) were used to obtained positive (CTRL +) and negative (CTRL -) methylation control, respectively.