Superscriptiii

LS180 cells were cultured for 16 h. Thereafter, the medium was changed to a fresh medium supplemented with MK-4 and Rif or control vehicle (EtOH) at 0.1% v/v following incubation for an appropriate length of time. The cells were washed twice with phosphate-buffered saline (PBS). Total RNA was isolated using ISOGEN reagent (Nippon Gene, Tokyo, Japan). Overall, 4 µg of total RNA was denatured with 2.5 µM oligo-dT primer (GE Healthcare, Tokyo, Japan) and 0.5 mM dNTP (GE Healthcare) at 65 °C for 5 min. The RNA was incubated in 20 µL reaction buffer [50 mM Tris-HCl at pH 8.3, 75 mM potassium chloride, 3 mM magnesium chloride, and 5 mM dithiothreitol (DTT)] containing 50 U of reverse transcriptase (SuperscriptⅢ, Invitrogen) and 20 U of RNase inhibitor (RNaseOUT, Invitrogen) at 50 °C for 60 min. An aliquot of synthesized cDNA was used as a template for following quantitative PCR. We used primers listed in Table 1 for amplification of the target cDNAs in the TB Green Premix Ex Taq solution (Takara Bio). Relative expression levels of mRNA were normalized to those of eukaryotic translation elongation factor 1 α1 (EEF1A1) mRNA.

Total RNA was extracted from INS-1D cells or mouse pancreatic islets using the RNeasy Mini Kit (Qiagen, Cologne, Germany). cDNA was prepared from 1 μg of total RNA using reverse transcriptase (SuperScript IIITM, Invitrogen) according to the manufacturer’s instructions. Quantitative PCR amplification was performed and analyzed using the TaqMan universal PCR master mix core reagent kit on StepOnePlus (Applied Biosystems, Foster, CA). The mRNA levels were evaluated with reference to beta actin (Actb) expression using the 2ΔΔCt method^{42 (link)}. TaqMan gene expression assay probes for rat Necab1 and Actb were obtained from Applied Biosystems (catalogue numbers Rn00574261_m1 and 4352931E, respectively, Applied Biosystems, Foster, IN). TaqMan gene expression assay probes for mouse Necab1, Actb, Insulin 1, and Insulin 2 were obtained from Applied Biosystems (Mm00724274_m1, Mm01205647_g1, Mm01278327_m1, and Mm03038438_m1, respectively).

Total RNA was extracted from cells using TRIzol Reagent (Invitrogen) according to manufacturer's instructions. Agarose gel electrophoresis and spectrophotometric measurements (NanoDrop ND-1000) were employed to estimate its concentration, quality and purity.
At least 0.5 μg of total RNA from each sample was retrotranscribed by SuperScriptIII^TM (Invitrogen) in the presence of random hexamers, according to the manufacturer's instructions. 1/100 of the resulting cDNA was used as substrate of any subsequent qPCR reaction. Limited to the intronless amplicons, prior to the retrotranscription, RNA preparations were treated by DNAseI (2U/μg of RNA) 1 h at 37°C, and processed by RNeasy Mini Kit (Qiagen). Next, negative control PCRs were run on RT⁻ cDNA preparations. In general, PCR reactions were performed by the SsoAdvanced SYBR Green Supermix^TM platform (Biorad), according to manufacturer's instructions. For each transcript under examination and each sample, cDNA was PCR-analyzed in technical triplicate, against absolute standards, and average results calculated. Averages were normalized against Gapdh and further normalized against controls. Experiments were performed at least in biological triplicate and analyzed by Student's t-test. Oligos were as in Supplementary Table S3.

The PnTlx sequence was retrieved from Pelagia noctiluca transcriptome. RNA extraction was performed on five days ephyra using the RNAqueous Total RNA Isolation Kit (Invitrogen AM1912) and cDNA was generated using the reverse transcriptase SuperScript III TM (Invitrogen). PCR products were cloned in pGEM-T Easy plasmid then transformed in competent cells. In situ hybridization probes were synthesized with T7 RNA polymerase. In situ hybridization was performed as previously described^{57 (link)} with some modifications. Ephyra (5 days post-fertilization), pre-ephyra (3dpf) and planula (2dpf) were generated from fertilized eggs collected from wild jellyfish caught in the bay of Villefranche-sur-Mer (France). Ephyrae were relaxed using 400 µM menthol. Ephyrae, pre-ephyrae and planulae were fixed on ice with a pre-chilled solution of 3.7% formaldehyde plus 0.4% glutaraldehyde in 1X PBS (Phosphate-Buffered Saline), for one hour. Before the acetylation step, embryos were treated for 20 min in 10 µg/ml of proteinase K (Fisher BioReagents™ BP1700-500) followed by two washes in 4 mg/ml of glycine to stop the action of proteinase K. Then embryos were post-fixed in 3.7% formaldehyde in 1X PBST. 5% of dextran was used in the hybridization buffer and SSC pH 4.7.

Total RNA was extracted from cells using TRIzol Reagent (Invitrogen) according to manufacturer’s instructions. Agarose gel electrophoresis and spectrophotometric measurements (NanoDrop ND-1000) were employed to estimate its concentration, quality and purity. RNA preparations were treated by TurboDNAseI kit (Gibco) 1 h at 37 °C. At least 0.5 μg of total RNA from each sample was retrotranscribed by SuperScriptIII^TM (Invitrogen) in the presence of random hexamers, according to the manufacturer’s instructions. 1/100 of the resulting cDNA was used as substrate of any subsequent qPCR reaction. Next, negative control PCRs were run on RT− cDNA preparations. In general, PCR reactions were performed by the SsoAdvanced SYBR Green Supermix^TM platform (Biorad), according to manufacturer’s instructions. For each transcript under examination and each sample, cDNA was PCR-analyzed in technical triplicate, against absolute standards, and average results calculated. Averages were normalized against Gapdh and further normalized against controls. Experiments were performed at least in biological triplicate and analyzed by Student’s t-test.

Quantitative real-time reverse transcription PCR (qRT-PCR) was used to quantify mRNA for gene expression profiling and validation of type II microarray analysis. Total RNA was reverse transcribed using Superscript III^TM (Invitrogen, UK) according to the manufacturer's protocol. Oligonucleotides for the target genes (Supplementary Table 1) were selected based on the genome of P. denitrificans PD1222 using Primer3 and ordered through Eurofins MWG^® Operon (DE). Gene expression was assessed with a Bio-Rad^®C1000 Thermal Cycler and CFX96 Real-time PCR detection system using SensiFAST™ SYBR^® Green Master Mix (Bioline, UK) according to the manufacturer's instructions The PCR assays were subjected to melt-curve analyses to identify/eliminate non-specific PCR products. Gene expression was normalised to the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) housekeeping gene using primers previously described (Sullivan et al., 2013 (link)) and relative expression ratios were calculated using primer efficiencies derived from standard curves as described previously (Pfaffl, 2001 (link)). The melt-curve analyses for GAPDH and fhp amplicons are presented in the supplementary data (Supplementary Fig. 1). All qRT-PCR experiments conformed to the Minimum Information of Quantitative Real-time PCR Experiment (MIQE) guidelines.

In each experimental session, aliquots of 6 × 10⁵ cells were processed for RNA extraction by Trizol^TM Reagent (ThermoFisher) according to manufacturer’s instructions. RNA preparations were treated by TURBO^TM DNase (2 U/μL) (Ambion^TM) 1 h at 37 °C. At least 0.75 μg of genomic DNA-free total RNA from each sample was retro-transcribed by SuperScriptIII^TM (Invitrogen) in the presence of random hexamers, according to manufacturer’s instructions. 1/100 of the resulting cDNA was used as substrate of any subsequent qPCR reaction. Limited to intronless amplicons, negative control amplifications were run on RT(-) RNA preparations. PCR reactions were performed by SsoAdvanced SYBR Green Supermix^TM (Biorad), according to manufacturer’s instructions. The oligonucleotides employed in this study are listed in Table S1. For each transcript under examination and each sample, cDNA was PCR-analyzed at least in technical triplicate and results averaged. When not otherwise specified, averages were further normalized against Gapdh. Experiments were performed at least in biological triplicates and analyzed by Student’s t test.