T3 rna polymerase

Total RNA (20 µg) extracted from sweet potato leaves was separated by electrophoresis using a 12% polyacrylamide gel containing 8 M urea (Amresco Inc., USA). Then the RNA gel was transferred to a Hybond-NX membrane (GE Healthcare, USA) and cross-linked by UV (Pall et al., 2007 (link)). For miR408 detection, the blotted membranes were hybridized with the radiolabeled gene-specific RNA probes, produced by in vitro transcription (Jeng et al., 1990 (link), 1992 (link)) using T3 RNA polymerase (Promega, Madison, WI, USA). The antisense sequence of miR408 fused with the T3 promoter was synthesized and annealed with the T3 top strand (Supplementary Table S2) as the DNA template for transcription to synthesize the miR408 RNA probe by T3 RNA polymerase (Promega). The procedures of pre-hybridization, hybridization, and washing were performed as previously described (Lin et al., 2012 (link)). The membrane was exposed to a Phosphorimager screen (Molecular Dynamics) for 3–4 d after washing, and then was scanned by Phosphorimager (Typhoon 9400). In addition, the membrane was stripped and re-hybridized with the radiolabeled 5.8S rRNA probe, produced by PCR with primers 5.8S rRNA-F/5.8S rRNA-R (Supplementary Table S2), and it served as an internal control for small RNA blot assays. Three independent experiments were performed for each sample.

We cloned all H3 cDNAs in the pβRN3P vector^{97 (link)}. This vector stabilizes RNA and improves their translation efficiency, while injected into Xenopus eggs. In addition, an HA-tag has been inserted in the C-terminal of H3. We obtained mRNAs by in vitro transcription of PCR-amplified fragments of the different pβRN3P vectors (forward: 5′-gtaaaacgacggccagt-3′ and reverse: 5′-ggaaacagctatgaccatga-3′). We transcribed mRNAs starting with 5 ng of PCR-amplified fragment, 10 μL Buffer 5×, 5 μL of dithiothreitol (DTT) 100 mM, 0.25 μL of bovine serum albumin (BSA) 10 mg/mL (NEB), 5 μL of ATP, CTP, UTP 10 mM, 1.65 μL of GTP 10 mM (Sigma-Aldrich), 3.35 μL of Me7GTP 10 mM (NEB), 2 μL of RNasin Plus RNase Inhibitor (Promega), and 50 μL H2O qsp. After 10 min on ice, we added 2 μL of T3 RNA Polymerase (Promega) to each sample,  incubated for 30 min at 37 °C, then added 0.5 μL of  fresh T3 RNA Polymerase and incubated for another 10 min at 37 °C. After DNA digestion with 2 μL RQ1 DNase (Promega) for  20 min at at 37 °C, we extracted mRNAs with phenol–chloroform and purified them through Sephadex G-50 Quick Spin Column for radiolabeled RNA purification (Sigma-Aldrich), previously equilibrated six times with 1 mL of TE 10 mM.

Candidate ORs which had less than 80% identity with all other ORs were chosen from the RNA-Seq data. Probes against their ORFs were prepared by the addition of the T3 start site to the 3’ end of the pCI plasmid primer followed by incorporation of digoxigenin (DIG) using T3 RNA polymerase (Promega) and alkaline degradation to get labeled probes of around 200 bp. Slides were prepared as described above and fixed in 4% PFA for 15 min and washed twice in PBS. They were acetylated in 1.2% triethanolamine and dropwise addition of acetic acid. They were washed in PBS for 5 min and prehybridized in buffer for an hour in large plates soaked in 5XSSC and 50% formamide at 58°C. 1 μl of the aforementioned labeled probe in 200 μl of the prehybridization buffer was pipetted on to the slides and covered with a layer of parafilm and kept overnight at 58°C. The slides were thoroughly washed in 5XSSC and then in 0.2XSSC twice for 30 min, 5 min in PBS and then blocked for an hour. The slides were stained with alkaline phosphatase conjugated antibody against DIG (Roche) for an hour and then kept in development buffer for 5 min before being subject to NBT-BCIP in development buffer overnight. Slides were then stained with bisbenzimide and mounted in Mowiol as described above.