Dntps mix

The ground tissue powder was suspended in 10 volumes/weight OTX Lysis Buffer, briefly needle-sonicated and centrifuged for 30 s at 14 000 rpm. Supernatants were collected, diluted at 1/750 in ultrapure water, and used for analysis. CL-qPCR was carried out following the protocol from Boos et al. (27 (link)), with minor modifications. For each tissue and each compound, a calibration curve was generated and run along with the samples. Mix composition for Chemical Ligation: 0.1 μl PS primer (10 μM stock), 0.1 μl BPS primer (10 μM primer), 1 μl Poly A (GE Healthcare #27411001, 1 mg/ml), 1 μl 10× Buffer with MgCl₂ (Roche #12032902001) and 5.8 μl ultrapure water, mixed with 2 μl diluted lysate. Chemical ligation was run for 1 h at 33°C. qPCR was run on a LC480 device (Roche). A qPCR mix containing 0.15 μl FW primer (10 μM stock), 0.15 μl RV primer (10 μM stock), 0.13 μl BHQ primer (28.28 μM stock), 0.15 μl dNTPs mix (ThermoFisher #10297018), 1 μl 10× Buffer with MgCl₂ (Roche #12032902001), 0.1 μl 10× FastTaq Polymerase (Roche #12032902001) and 6.32 μl ultrapure water was prepared and mixed with 2 μl of chemical ligation reaction mixture. qPCR program was as follows: 10 min, 95°C, 50 cycles (3 s/95°C – 30 s/55°C – 10 s/72°C). Concentrations in tissues were obtained through interpolation from the calibration curves and expressed in ng oligonucleotide/mg tissue for each mouse.

DNA isolates from each strain were then used as a template for PCR targeting bacterial 16S rRNA genes. PCR was performed in a 25-μl volume consisting of 2.5 μl of 10× DreamTaq green buffer (Thermo Fisher Scientific, Waltham, MA, USA), 0.5 μl of deoxynucleoside triphosphates (dNTPs) mix (Thermo Fisher Scientific, Waltham, MA, USA), 0.5 μl of each of two universal bacterial primers, i.e., 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-GGTTACCTTGTTACGACTT-3′) in 10 pmol μl⁻¹ concentration, 0.1 μl Taq DNA polymerase (Qiagen, Hilden, Germany), 1 μl of template DNA, and 19.9 μl of distilled water. For difficult templates (resulting in a small amount of expected product), PCR was repeated using Taq PCR core kit (Qiagen, Hilden, Germany) using the same template as before. Reaction was performed in a 50-μl volume consisting of 5 μl of 10× CoralLoad PCR buffer, 10 μl of 5× Q-solution, 1 μl of dNTPs mix (Thermo Fisher Scientific, Waltham, MA, USA), 2.5 μl of each primer, 0.25 μl of Taq DNA polymerase, at least 3 μl of template DNA (depending on concentration), and distilled water up to 50 μl. PCR was performed as follows: 3 min in 94°C for initial denaturation, 35 cycles of 30 s in 94°C, 30 s in 53°C, 1 min in 72°C for annealing, and 10 min in 72°C for final elongation. Presence of the PCR product was confirmed on 1% agarose gel and then purified and cloned.

Following DNase I treatment, 1 µL of Random Hexamers (50 µM) and 1 µL of dNTPs mix (10 nM each) (Thermo Fisher Scientific) were added and samples were incubated at 65°C for 5 min followed by 5 min at 4°C. After that, 4 µL of 5x SSIV Buffer (Invitrogen), 1 µL of dithiothreitol (DTT; 100 mM; Invitrogen), 0.5 µL of Ribolock Recombinant RNase Inhibitor (40 U/µL; Thermo Fisher Scientific), and 0.5 µL of Superscript IV (SSIV) Reverse Transcriptase (200 U/µL; Invitrogen) were added to each sample. As a negative control to detect genomic DNA, the same mixtures were prepared for each sample, but without the addition of SSIV RT (-RT). Samples were incubated for 10 min at 23°C, 10 min at 55°C, and finally, 10 min at 80°C to inactivate the enzyme. Samples were stored at −20°C until further use.

The colony displaying zone of hydrolysis in LB agar tributyrin plates was cultured overnight in 5 ml LB at 37 °C in shaker (220 rpm). Genomic DNA was extracted from overnight grown culture employing standard phenol–chloroform method. Purified DNA was used for PCR amplification of the 16S rRNA gene, using universal primers: 27F (5′-AGAGTTTGATCMTGGCTCAG-3′) and 1642R (5′-CGGYTACCTTGTTACGAC-3′), respectively. The following thermal cycling conditions were used to amplify the 16S rRNA gene, 94 °C for 4 min, followed by 30 cycles of 94 °C for 50 s, 55 °C for 50 s, 72 °C for 2 min, with final extension of 7 min at 72 °C. The 25 µl PCR mix contained 1 µl (0.1 µg) DNA, 1 μl each for ward and reverse primers (2.5 µM), 200–400 µM dNTPs mix (Thermo Scientific), 1U Taq DNA polymerase (Thermo Scientific) and 1× reaction buffer (Thermo Scientific). PCR-amplified product was checked on a 1.5 % agarose gel, and purified from gel employing XcelGen DNA Gel/PCR Purification kit (Xcelris genomics). The amplified product was cloned in pGEM-T easy vector (Promega, USA) as per manufacturer’s instruction. The recombinant plasmid DNA was extracted from white colonies employing Hi Yield™ Plasmid DNA Mini Kit (Real Genomics) and sequenced using M13 forward and reverses primer.

A single PCR mix included Phusion HF Buffer (Thermo Scientific), 2.5 mM dNTPs mix (Thermo Scientific), 1.5 mM MgCl₂ (Thermo Scientific), PfuX7 DNA polymerase (homemade), primers (0.5 μM each), and 1 ng/µL DNA template. Target DNA was PCR-amplified by 30 cycles of denaturing (94 °C for 30 s), annealing (5 °C or more lower then T_m for 30 s), and extending (68 °C for 1 min per 1 kbp). Amplifications were confirmed by 1 or 2% agarose gel electrophoretic analyses. The list of primers and their sequences is provided in Supplementary Table 8.

cDNA was generated using SuperScript III reverse transcriptase (cat. no. 18080-044, Invitrogen, Thermo Scientific), random hexamer primers (cat. no. SO142, Thermo Scientific), 10 nM dNTPs Mix (cat. no. R0192, Thermo Scientific) and RNaseOUT to inhibit ribonucleases (cat. no. 10777-019, Invitrogen) in accordance with the manufacturer's instructions. qPCR assays were performed in a 7900HT Fast Real-Time PCR System (Applied Biosystems, Thermo Scientific) on MicroAmp Optical 384-well plates (Applied Biosystems). Each assay contained 2X SYBR Green PCR Master Mix (cat. no. 4309155, Applied Biosystems), 250 nM each of the forward and reverse primers (synthesized by IDT, Leuven, Belgium) (Supplementary Table S1) and 12.5 ng cDNA. The relative levels of mRNA were quantified by the comparative Cq procedure (ΔΔCq) and each value then related to the corresponding mock condition as well as to the RNA values of the references β-actin and 18S ribosomal RNA.