C1000 touch

Transcript levels of IRF1, IRF2, IRF3, IRF5, IRF7, IRF9, IFNAR1, IFIT1, MX1, IFN-β, RANTES (CCL5), and TNFAIP3 (A20) genes were measured by quantitative RT-PCR. According to the manufacturer’s protocol total RNA was isolated from cell lysates using the Monarch Total RNA Miniprep Kit (T2010S, NEB). Reverse transcription was performed using the High-Capacity cDNA Reverse Transcription Kit (43-688-14, Applied Biosystems) following the manufacturer’s protocol in a thermal cycler (C1000 Touch™, Bio-Rad). Gene specific primers (see Table 3) with exon-exon junction overlap designed with PrimerBlast [32 (link)] and iTaq Universal SYBR Green Supermix (17251525, Bio-Rad, Germany) were used to perform quantitative RT-PCR. Relative transcript levels were determined using only the ΔC_T (relative to GAPDH), and not normalizing to mock / unstimulated controls as in the ∆∆C_T method. This was favorable as the expression of many genes of interest (e.g., IFNB1) is minute and not reliably measurable in the absence of stimulation, and normalization to such unreliable background values close to zero would introduce a high degree of artificial noise.

RNA extraction from the liver of juveniles (n = 9 from each experimental group) was carried out using a similar protocol described above for the qPCR. Absolute gene expression analysis was performed with Droplet Digital PCR (ddPCR) using Bio-rad QX200 (Hercules, CA, USA) systems, using the same cDNA obtained as above. Samples were prepared using the workflow provided by the manufacturer. In summary, for each gene, master mixes were prepared using 10 μL EvaGreen super mix (Bio-rad, Hercules, CA, USA), 0.2 μL F primer, 0.2 μL R primer, 7.6 μL MilQ water, and 2 μL cDNA (approx. 20 ng cDNA). Primers, Genbank accession numbers and reference articles for sequences of target (lpl, ppara, elovl6, fads2, cox2, cpt1) and housekeeping gene (ß-act) were provided in Supplementary Table S1. Droplets were generated using droplet generator Bio-rad QX200 (Hercules, CA, USA) and were transferred to 96-well microplates for PCR in a thermal cycler (Bio-rad C1000 Touch, Hercules, CA, USA). Following PCR amplification, droplets were read in a droplet reader (Bio-rad QX200, Hercules, CA, USA) to determine absolute gene expression. Readings lower than 12000 droplets were not used for the gene expression.

The denaturation of the bacterial genome to create genomic ssDNA was achieved by heating genomic samples at 95 ${}^{°}$ C for 10 min in a Thermal Cycler (Bio-Rad®, C1000 Touch, U.S.A.). Subsequently, samples could either be used immediately or be refrigerated to $-$ 20 ${}^{°}$ C for future use.
50 μL of MMV-PS was added to 50 μL of genomic DNA solution (g/L) followed by the addition of 10 μL of TE buffer containing 6 mM NaCl. 5 μL of DAPI was added to the mixtures, and the whole sample was incubated for 10 min with mild shaking. Following that, 20 μL of the mixture was loaded slowly into a poly-dimethylsiloxane (PDMS) chamber (a hollow PDMS cylinder sandwiched by two glass slides.) Both the PDMS and the glass were washed with 75% ethanol and pretreated with plasma to prepare them for use in a confocal fluorescence microscope.

Total RNA extraction from peripheral blood cells and liver were made using a similar protocol described above for the qPCR. Absolute gene expression of PBCs and liver fads2 analysis were performed using Digital Droplet PCR (ddPCR) (Bio-rad QX200, Hercules, CA, USA) systems, by using cDNA obtained as mentioned in Section 2.3. Sample preparation for ddPCR was carried out as per the manufacturer’s protocol. The master mixes for fads2 gene were prepared including 10 μL EvaGreen super mix (Bio-rad, Hercules, CA, USA), 0.2 μL F primer (10 pmol/μL), 0.2 μL R primer (10 pmol/μL), 7.6 μL MilQ water, and 2 μL cDNA. Then, droplets were generated using droplet generator Bio-rad QX200 (Hercules, CA, USA) and the droplets were transferred to 96 well microplates for PCR in a thermal cycler (Bio-rad C1000 Touch, Hercules, CA, USA). After PCR amplifications, droplets were measured with a droplet reader (Bio-rad QX200, Hercules, CA, USA) to determine absolute gene expression of fads2 gene. The samples with less than 12,000 droplets were not used for the gene expression study. The fads2 gene expression analysis was performed in two replicates for each sample and values were expressed as mRNA copies/µL [31 (link),33 (link)].

CETSA was performed as described by Molina et al.^{44 (link)}. Briefly, PC3 cells were treated with 10 μM JA2131 or an equivalent volume of DMSO for 2 h, washed three times with PBS and extracted for total cell lysate. Ten percent glycerol was added to the samples before subjecting to heat. Gradient thermal cycler (C1000 Touch, Bio-Rad) was used to heat samples at 42, 57, 66, 72, 77, and 85 °C for 3 min and chilled on ice for 5 min. Then, samples were centrifuged at 14,000 rpm for 20 min at 4 °C. Supernatants were carefully removed and analyzed by western blotting analysis.