Fastdigest enzymes

All restriction enzymes used were fast digest enzymes (ThermoFisher Scientific, United Kingdom), the ligase used throughout this work was the Rapid DNA Ligation Kit (ThermoFisher Scientific, United Kingdom). Plasmids were transformed into chemically competent E. coli HB101 according to supplier’s instructions (New England Biolabs, United Kingdom). Plasmids are listed in Supplementary Table 1 and primers and synthetic DNA fragments are listed in Supplementary Table 2.

Genomic DNA (5–40 ng) or cfDNA (from ∼1 ml of whole blood) was digested using multiple restriction enzymes [Set1: AlwNI and Alw26I; Set2: EarI and NcoI; SetKC: EarI and NmuCI (FastDigest enzymes, Thermo Scientific, MA, USA); Supplementary Table S1]. The ligation of adaptors with N₁₂ barcode sequence tags was performed using Escherichia coli DNA ligase (Takara Bio, Shiga, Japan). The ligation products were purified twice with a 1.2× volume of AMPureXP beads (Beckman Coulter, CA, USA). Linear amplification of the purified products was performed with a region-specific primer mixture (Supplementary Tables S2 and S3) and Q5 Hot Start High-Fidelity DNA Polymerase (NEB) by 10 thermal cycles. The purified linear amplification products were amplified with the PGM/Proton primers (Supplementary Table S2) and Platinum Taq High Fidelity (Life Technologies). The amplification products were purified with AMPureXP beads or agarose gel electrophoresis with a MinElute Gel Extraction Kit (Qiagen). Further details and library construction for experiments with double strand labeling are provided in Supplementary Methods.

The open reading frames (ORFs) of cdaA, pde1 and pde2 as well as the fragments encoding amino acids 94–285 and 103–285 of CdaA and amino acids 53–657 of Pde1 were amplified from genomic DNA of S. mitis CCUG 31611 using primers listed in Table S1. Amplicons were cloned into the pET28a(+)KpnI-mut protein expression vector between the KpnI and HindIII sites, using FastDigest^™ enzymes (ThermoFisher Scientific, Waltham, MA, USA) and the Rapid DNA Ligation kit (ThermoFisher Scientific). The pET28(+)KpnI-mut vector was constructed to simplify sub-cloning of the relevant genes between vectors. Constructs were verified by Sanger sequencing at Eurofins Genomics (Ebersberg, Germany). E. coli BL21 (DE3) cells harbouring constructs were grown in LB with 50 mg/L kanamycin at 37 °C and 190 RPM until an OD₆₀₀ of 0.4–0.5. Protein expression was induced by addition of isopropyl β-D-1-thiogalactopyranoside (IPTG; MilliporeSigma) at a final concentration of 50 µM. Protein expression was maintained for 3 h at 37 °C, 190 RPM.

FastDigest enzymes (Thermo Scientific, Waltham, MA, USA) were used for all DNA restriction digestions. T4 DNA Ligase (Thermo Scientific EL0014) was used for ligation.

The total RNA from mouse cells was isolated using the NucleoSpin RNA Kit according to the manufacturer’s instructions (Macherey Nagel). The total RNA from mouse tissues was isolated using peqGold TriFast^TM (peqlab, Erlangen, Germany) according to the manufacturer’s instructions. The analysis was performed according to a method published originally by Okunade et al. [35 (link)] and recently adapted by our group [46 (link)]. This strategy is based on first simultaneously amplifying Pmca1 and Pmca4 cDNA fragments, followed by a gene-specific restriction digest of the PCR products. The first strand cDNA was prepared using the RevertAid H Minus First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Baltics UAB, Vilnius, Lithuania). Briefly, 375 ng of total RNA were reverse transcribed using random primers, followed by a PCR using the primers published by Okunade et al. [35 (link)]. These primers allow for the efficient amplification of a 1 kb region of Pmca1 and 4, but not Pmca2 and 3. Restriction digests of the PCR products were performed with either ClaI (C) or BglI (B) or both (CB) (FastDigest enzymes, Thermo Fisher Scientific) or neither (uncut, U), yielding fragments of 400 bp and 600 bp characteristic for Pmca1 (ClaI cut) or two ~500 bp fragments for Pmca4 (BglI cut) as shown in the scheme in Figure 1E.

Strain cultivation and DNA manipulation followed standard methods. DNA sequencing and all primers were purchased from Tsingke Biotechnology Co., Ltd. (China). Primers used for PCR and site-directed mutagenesis are listed in Supplementary Table 1. All Fast Digest enzymes were from Thermo Scientific. DNA polymerases were purchased from Invitrogen Trading Co., Ltd. (China). All the other chemicals and reagents were purchased from commercial suppliers and used without further purification. ClustalX was used in protein sequence alignments.