Mycycler thermal cycler system

The cDNA synthesis from corresponding RNA was done using the QuantiNova™ Reverse Transcription Kit (Qiagen®, Germany). Freshly filtered double-distilled water, RNA at 1000 ng/reaction, Reverse Transcription Reaction Mix, and Reverse Transcription Enzyme were added to a 200 μL PCR tube (Axygen™, Fisher Scientific®, USA). The tubes were then placed in a thermocycler (MyCycler™ Thermal Cycler System, Bio-Rad Laboratories Inc., USA) that was run for one cycle of 5 min at 25°C, 30 min at 42°C, and 5 min at 85°C. Finally, the samples were held at 16°C until collection. The cDNA was stored at -20°C.

Two or more PCR tubes were filled with high titer stock of bacteriophages suspended in DPBS. The tubes for the thermal incubation condition were placed in a Bio Rad MyCycler thermal cycler system, and were kept at a constant temperature between 60 and 70 ${}^{\circ }$ C for 1 h, control group tubes were kept at 25 ${}^{\circ }$ C for 1 h. The titer of each group was then quantified using the aforementioned spot-test protocol, and the surviving fraction of active bacteriophages was found by dividing the titer of thermal selection groups by the titer of the low temperature control group. For initial thermal stability assays (Fig. 2a), an initial titer of $1\times {10}^{10}$ PFU/mL was used. In time-dependent degradation assays (Fig. 2b), an initial titer of $1\times {10}^8$ PFU/mL was used.

PCR conditions (annealing temperature and numbers of PCR cycles) were optimized to identify 5 of the 7 target species and genera using the primer sets shown in Table 1. Amplification was performed in a 25 μl mixture containing Mg²⁺, dNTPs, and recombinant Taq DNA Polymerase for routine PCR of fragments up to 5 kb (Invitrogen, USA), 10 μM forward and reverse primers and 2 μl bacterial DNA extract in a thermal cycler (MyCycler™ Thermal Cycler system, BioRad). PCR was carried out using the following conditions: an initial denaturation step for 4 min at 94 °C, with 45 cycles of 30 s at 95 °C, 1 min at 58 °C and 30 s at 72 °C, followed by 5 min at 72 °C. Agarose gel (Sigma, USA) was prepared at a concentration of 1.5% (w/v) in 60 ml Tris-Borate Buffer (TBE). One μl of 10 μg/ml ethidium bromide (Sigma, USA) was incorporated into the gel to make a final concentration of 0.5 μg/ml and electrophoresed at 90 V for 60 min. The DNA bands were visualized using a gel documentation system (ChemiDoc XRS, Bio-Rad, USA) under ultraviolet (UV) illumination.

PDGF-B retention motif KO was assessed in BMDMs through DNA genotyping. A master mix containing REDExtract-N-Amp PCR ReadyMix (R4775, Sigma-Aldrich) and specific forward and reverse primers (10 µM, Supplementary Material, Table S2) was added per DNA sample (≥100 ng) and separately for both the Pdgfb^WT and Pdgfb^ret gene. Subsequently, PCR was performed in a thermal cycler (MyCycler Thermal Cycler System, Bio-Rad, Hercules, CA, USA). An agarose gel was casted using agarose, 0.5×TBE buffer (tris-borate-EDTA buffer, 45 mM tris-borate 1 mM EDTA in dH₂O) and SYBR Safe DNA Gel stain (1:35,000 dilution, S33102, Invitrogen). The resulting PCR samples and a DNA ladder (GeneRuler 100 bp Plus DNA ladder, SM0321, Thermo Fisher Scientific) were loaded and electrophoresis was performed for 30 min on 120 V (Powerpac 300, Bio-Rad).