Rq1 rnase free dnase treatment

Total RNA was extracted using Trizol Reagent (Invitrogen Ambion) according to the manufacturer’s recommendations, followed by a RQ1 RNase-free DNase treatment (Promega) and a cDNA synthesis from 1 μg RNA using miScript II RT kit (Qiagen). Quantitative real-time PCR was performed in triplicate with diluted cDNA (either 1/1000 or 1/10000, for minor or major spliceosomal snRNAs respectively) using SYBR Green ROX Mix (Thermo Scientific) with an Applied Biosystems 7500 fast system. Three independent experiments were carried out for each condition. The snRNA, 5 S and 5.8 S primers have been previously described^{22 (link),25 (link)}. The normalized expression levels were calculated according to the ΔΔCt method to establish the relative expression ratio between the vehicle-treated control mice and the other groups of mice. The analyses of the SMN2 exon 7, SNAP25 exon 5 and AGRN Z’exon were performed as described^{26 (link),39 (link),77 (link)}. Additional primers have been designed. All primers are listed in Table S5 in the supplemental material.

EcoRI-linearized FHV_T_Rluc and FHV_T_minus and XhoI-linearized pEAV221Δ [34 (link)] plasmids were used as templates for in vitro transcription. Transcripts of FHV_T_Rluc and FHV_T_minus corresponding to capture probes for minus and plus strands, respectively, and a transcript containing nucleotides 1-2042 of equine arteritis virus (EAV) genome were prepared using mMESSAGE mMACHINE T7 Transcription Kit (Ambion) according to the manufacturer’s instructions. In vitro transcription reactions to prepare ³²P-labeled transcripts of FHV_T_Rluc and FHV_T_minus consisted of 750 ng of the respective linearized plasmid, transcription buffer (Promega), 800 U/mL T7 RNA polymerase (Promega), 1000 U/mL RiboLock (Thermo Fisher Scientific), 5 mM DTT, 1 mM ATP, UTP, GTP and CTP, and 0.133 μM α-³²P-CTP (20 μCi) (Perkin Elmer, Ayer Rajah, Singapore). The reactions were incubated for 2 h at 37 °C followed by RQ1 RNase-free DNase treatment (40 U/mL; Promega) for 35 min at 37 °C and inactivation by RQ1 DNase STOP solution (Promega) for 10 min at 65 °C. Unincorporated label was removed using RNase-free Micro Bio-Spin P-30 Gel Columns (Bio-Rad). Hybridization with the ³²P-labeled RNA transcripts and IVRA products was performed as described [34 (link)].

Two-week-old seedlings of Shen Nong 9816 (SN9816) grown in 14 h light and 10 h dark were collected. The harvested seedlings were treated with 0 and 250 mM NaCl; 0 and 100 μM ABA; 0 and 100 μM JA for 6 h. Two biological replications were performed for each treatment. Total RNA of the samples was isolated using Takara RNAiso Plus (Takara Bio Inc., Otsu, Japan). The contimanate DNA was removed by using RQ1 RNase-Free DNase treatment (Promega, Madison, WI, USA). Then, 3 μg of treated RNA was used to synthesize the first-strand cDNA with the RevertAid First Strand cDNA Synthesis Kit (Fermentas, Waltham, MA, USA). SYBR Premix Ex Taq (Takara Bio Inc., Shiga, Japan) and a Q5 Real-Time PCR Instrument (Applied Biosystems, Waltham, MA, USA) were used for qRT-PCR [95 (link),96 (link)]. Primers used for qRT-PCR are listed (Table S2).
The Mircroarray data are from the Rice Expression Profile Database (RiceXPro, http://ricexpro.dna.affrc.go.jp/, accessed on 13 May 2021). Four-leaf-old seedlings of rice were treated with 50 μM ABA and 100 μM MeJA for 0, 1, 3, 6, and 12 h; two biological replicates were performed. The shoot of the treated seedlings was used for microarray analysis. The signal intensity detected during hybridization was used for drawing heatmap with TBtools.

Cells were detached with Trypsin-EDTA from their culture dish, centrifuged for 1 min at 2000 rpm in a table biofuge (Heraeus). To extract total RNA, EZ-RNA Kit (Biological Industries, Israel) was used according to the manufacturer instructions. After RNase free RQ1 DNase treatment (Promega) total RNA was subjected to sodium periodate treatment as outlined by Tzadok et al., 2013 [in ref. 10 (link)].
Nuclear RNA was isolated by perforating the cells with 0.1% digitonin in Hank’s Balanced Salt Solution (HBSS) and 1mM PMSF. Following, cell ghosts were collected by a low speed spin. The pellet was further subjected to RNA extraction, with the EZ-RNA Kit (Biological Industries).