Ssoadvanced universal sybr green master mix

Takara Ex Taq Kit (Takara Bio Inc., Japan) was used to perform end-point PCR according to manufacturer’s instructions. Primers used are listed in Supplementary Table 5.
Conventional quantification of gene or miRNA expression was assessed by qRT-PCR using No AmpErase TaqMan Universal PCR Master Mix (Thermo Fisher Scientific Inc., Waltham, MA, USA) and specific TaqMan gene-expression assays (Thermo Fisher Scientific Inc., Waltham, MA, USA), as listed in Supplementary Table 4. Amplifications were run on the 7900HT Fast Real-Time PCR System. Data were analyzed by SDS relative quantity (RQ) Manager 1.2.1 software (Thermo Fisher Scientific Inc., Waltham, MA, USA) and reported as RQ or −ΔΔCt with respect to a calibrator sample using the comparative Ct (ΔΔCt) method. GAPDH/SNORA74A and RNU48 were used as normalizers in gene and miRNA expression studies, respectively. If not otherwise specified, MIR205HG/LEADR expression was measured using an assay covering ex-2/3 boundary, which detects all possible transcripts.
Enrichment of DNA regions in ChIP and ChIRP experiments was evaluated through SYBR green qPCR run on a CFX Connect Real-time PCR instrument, using SsoAdvanced Universal Sybr Green Master mix (Bio-Rad, Hercules, CA, USA) and primers listed in Supplementary Table 5.

Total RNA was extracted from cultured cells using an RNeasy Microkit, including DNase incubation, according to the instructions of the manufacturer (Qiagen). One microgram of total RNA was reverse transcribed to complementary DNA (cDNA) using 0.5 μg of oligo(dT)_15‐18 primers, 1.0 mM dNTPs, 1× Reaction Buffer, 20 units of RiboLock, and 200 units of RevertAid reverse transcriptase (all from ThermoFisher Scientific) in a total volume of 20 μl per reaction. The cDNA product was diluted 10‐fold in water and used at this concentration for qPCR. Qualitative PCR was performed using SsoAdvanced Universal SYBR Green qPCR Master Mix (Bio‐Rad), with primers at a final concentration of 500 nM from a 10 μM stock. Diluted cDNA (2.5 μl) was used per reaction, and all reactions were performed in triplicate in a total volume of 10 μl. Primer sequences are shown in Supplementary Table 1, available on the Arthritis & Rheumatology web site at http://onlinelibrary.wiley.com/doi/10.1002/art.40659/abstract. A 2‐step qPCR cycle with a Bio‐Rad iCycler qPCR machine was used for target amplification, according to the instructions of the manufacturer for SSoAdvanced Universal SYBR Green Master Mix, and CFX Manager was used for analysis.

Total RNA was prepared from co-incubation assays of V. cholerae with T. pyriformis and A. castellanii in 24-well plates. Bacterial cells from within protozoa were collected as described above and RNA was extracted by lysozyme digestion followed by use of the Aurum Total RNA mini kit (Bio-Rad, Hercules, CA, USA) according to the manufacturer’s instructions. The concentration of RNA was measured by spectrophotometry (NanoDrop ND-1000; NanoDrop Technologies). Complementary DNA (cDNA) was prepared from 400 ng RNA from each sample by iScript Reverse Transcription (Bio-Rad, Hercules, CA, USA) following the manufacturer’s instructions. Quantitative reverse-transcriptase PCR (qRT-PCR) was conducted using SsoAdvanced Universal SYBR Green Master Mix (Bio-Rad, Hercules, CA, USA) and a QuantStudio 6 Flex Real-Time PCR System using V. cholerae integron-integrase specific primers (intIA-1F/intIA-1R) (Supplementary Table 2). Transcription of intIA was determined relative to the transcription of gyrA (gyrA_F/gyrA_R) (Supplementary Table 2) using the comparative Ct (ΔΔCt) method.

RNA was extracted with the RNeasy Plus Mini Kit (Qiagen, Hilden, Germany) from untreated embryos and those treated with PCP at 24 and 48 hours. Genomic DNA was removed from samples with gDNA eliminator mini spin columns (Qiagen, Hilden, Germany). iScript (Bio-Rad, Hercules, CA) was used to generate cDNA from each sample. Each cDNA sample was made by pooling ten random whole embryos per condition. SsoAdvanced Universal SYBR green mastermix (Bio-Rad, Hercules, CA) was used to amplify genes, with an Eppendorf realplex2 (Eppendorf, Hamburg, Germany) machine using primers for ef1α [21 (link)], runx1 [21 (link)], cmyb [21 (link)], and itga2b (commonly referred to as cd41) [21 (link)]. All PCR primers were validated previously and designed to span exons. Data were analyzed for relative expression change with ef1α as the reference gene. ΔΔCt was calculated by comparing the expression of the treated embryos to control embryos and to the reference gene, ef1α.

For semiquantitative and real-time reverse-transcription (RT)-PCR, RNA was extracted using the methods described above, and single-strand cDNA libraries were synthesized with DNase I-treated RNA using Superscript III reverse transcriptase kit (Invitrogen) or, in the real-time experiments, iScript cDNA Synthesis Kit (Bio-Rad Laboratories, Hercules, CA). For semiquantitative RT-PCR, conditions for PCR reactions were optimized for each gene such that the endpoint of each PCR reaction was in the linear range of amplifications. Transcript-level quantification analyses were measured by analyzing pixel intensity of the bands using Image J (NIH). For quantitative real-time RT-PCR, cDNA samples were analyzed using a CFX96 Real-Time System (Bio-Rad Laboratories), with each 20 μl reaction containing 10 μl of SsoAdvanced Universal SYBR Green Mastermix (Bio-Rad Laboratories), 2 ng of cDNA, and RT primers designed using PrimerQuest (Integrated DNA Technologies, Coralville, IA) at a final concentration of 300 nM. Before RT-PCR experiments, primer efficiencies were determined from dilution curves (1:10) using the formula: E = 10^–1/slope (Pfaffl, 2001 (link)). Fold-change was determined using the ΔΔCt method (Livak and Schmittgen, 2001 (link)). Primers used in the RT-PCR experiments are listed in Table 7.