Superscript 3 kit

Total RNA from tissues was isolated using Trizol reagent (Invitrogen). The reverse transcriptase reaction (RT) was typically performed with 2 µg RNA, the Superscript III Kit, and random primers (all from ThermoFisher Scientific, Waltham, MA), followed by amplification with the 2X PANGEA-Long PCR Master Mix (Canvax Biotech, Córdoba, Spain). For conventional PCR mouse Insr was amplified using the following primers: sense, 5′-GGCCAGTGAGTGCTGCTCATGC-3′ (inside exon 10); antisense, 5′-TGTGGTGGCTGTCACATTCC-3’ (inside exon 12). Mouse β-actin was amplified using the following primers: sense, 5′-AAGGCCAACCGTGAAAAGAT-3’; antisense, 5′-GTGGTACGACCAGAGGCATAC-3’. For quantitative (q)PCR before performing the RT reaction, total RNA was treated with DNAse I (ThermoFisher Scientific) to eliminate potential contaminating DNA. RT was performed with 1 µg RNA and also with the Superscript III Kit and random primers (ThermoFisher). qPCR was performed with the ABI Prism 7900HT Sequence Detection System using Taq-Man Universal PCR Master Mix, no-AmpEThrase UNG, and Taqman assays (all from ThermoFisher): for Rps6 (Mm02342456_g1); for Insr (Mm01211875_m1) and for Tbp (Mm01277042_m1).

Total RNA from cultured cells were extracted using the RNeasy kit (Qiagen), and cDNA was generated from RNA using the Superscript III kit and oligo(dT) primers (Invitrogen). For generation of cDNA from mouse liver samples, total RNA was extracted from mouse liver samples using the RNeasy kit (Qiagen) and a dounce homogenizer (Ambion) according to manufacturer’s instructions. To generate cDNA from mouse liver RNA, oligo(dT) and random hexamers were used in equimolar concentration with the Superscript III kit (Invitrogen). Quantitative real-time PCR was performed using SYBR Green reagents in a StepOnePlus system (Applied Biosystems). Please refer to Supplementary Table 5 for a list of all primers for real-time PCR used in this study.

DNA‐free RNA was used for glnA cDNA synthesis using Superscript III kit (Invitrogen) and gene specific priming. The initial RT mixture containing 3 μl water, 1 μl reverse primer GS1_new (10 μM), 1 μl dNTP's (10 mM each) and 5 μl template was incubated at 65°C for 5 min and quickly transferred on ice for 1 min. A second mix composed of 4 μl 5X first‐strand buffer, 1 μl 0.1 mM dithiothreitol (DTT) and 1 μl SuperScript III (200 units μl⁻¹) was added and the resulting mixture was incubated at 55°C for 50 min and then at 72°C for 15 min. The primers and PCR conditions for the amplification of glnA targets from cDNA were similar to those used for DNA.
For 16S rRNA and amoA genes, Superscript III kit (Invitrogen) and random hexamers priming was used. The initial RT mixture containing 3 μl water, 1 μl random hexamer (50 μM), 1 μl dNTP's (10 mM each) and 5 μl template was incubated at 65°C for 5 min and quickly transferred to ice for 1 min. A second mix composed of 4 μl 5X first‐strand buffer, 1 μl 0.1 mM dithiothreitol (DTT) and 1 μl SuperScript III (200 units μl⁻¹) and 1 μl RNase inhibitor (40 U μl⁻¹) was added and the resulting mixture was incubated at 25°C for 5 min, 55°C for 50 min and then at 72°C for 15 min.