Rq1 dnase

Trizol was used to extract total RNA (Ambion, 15596-018). Total RNA was treated with RQ1 DNase (Promega) to remove DNA. A SmartSpec Plus™ spectrophotometer (Bio-Rad) was used to determine the quality and quantity of RNA by measuring the absorbance at 260 nm/280 nm (A₂₆₀/A₂₈₀). Electrophoresis with a 1.5% agarose gel was used to confirm RNA integrity.
RNA-seq libraries were created for each sample using 1 μg of total RNA. mRNAs were captured using VAHTS mRNA capture Beads (Vazyme, N401). The purified RNA was treated with RQ1 DNase (Promega) for the removal of DNA before being utilized for the directional RNA-seq library created using the KAPA Stranded mRNA-Seq Kit for Illumina^® Platforms (KK8544). Polyadenylated mRNAs were purified and fragmented. Fragmented mRNAs were then converted into double-stranded cDNA. Following end repair and A tailing, the DNAs were ligated to a Diluted Roche Adaptor (KK8726). After purification of the ligation product and size fractioning to 300–500 bp, the ligated products were amplified, purified, quantified, and stored at –80°C before sequencing. The strand marked with dUTP (i.e., the second cDNA strand) is not usually amplified, allowing for strand-specific sequencing.
The manufacturer’s instructions were followed for high-throughput sequencing and an Illumina NovaSeq 6000 system was used for 150 nt paired-end sequencing of the cDNA libraries.

RNA was isolated from 10-day-old seedlings. First-strand cDNAs were synthesized using RNA treated with RNase-free RQ1 DNases (Promega, United States) with SuperScript III-MMLV reverse transcriptase (Invitrogen, United States) and the oligo(dT)₁₅ primer. Quantitative RT-PCR was performed using SYBR Green PCR Master Mix (Applied Biosystems, United States). UBQ5 (At3g62250) served as the control. The primers used are shown in Supplementary Table 1.

Total RNA was extracted from 100 mg N. benthamiana leaf tissue using TRI Reagent Solution (Invitrogen) homogenized using TissueLyser II (Qiagen). Following homogenization, chlorophorm was added (200 ul/1 ml of TRI reagent) and centrifuged at 12,000 g for 20 minutes at 4 °C, after which, the aqueous phase was transformed into equivolume of isopropanol, and centrifuged at 12,000 g for 20 minutes at 4 °C. Pellet RNA was washed 3x with 75% ethanol before being resuspended in DNase/RNAse-free distilled H₂O. Extracted RNA was treated with RQ1 Dnase (Promega) at 37 °C for 1 hour.

Wild‐type P. ananatis PA13 was grown in LB medium to exponential growth phase (12 h after inoculation); total RNA was isolated using an RNeasy Mini Kit according to the supplier's instructions (Qiagen); the RNA samples were treated with RQ1 DNase (Promega) to remove any contaminating DNA. RT‐PCR was performed according to a previous report (Kim et al., 2004) as follows. Total RNA from P. ananatis PA13 was reverse transcribed into cDNA using M‐MLV reverse transcriptase as described by the manufacturer (Promega) at 50°C for 1 h, followed by 5 min at 75°C. Next, PCR was performed using a T100 Thermal Cycler (Bio‐Rad) under the following conditions: 96°C for 2 min, followed by 40 cycles of 96°C for 1 min, 50°C for 1 min, and 72°C for 1 min. The following primers were used for RT reactions, RT1 (crtB), and RT2 (crtZ). The following PCR primers were used: PCR1f and PCR1r; PCR2f and PCR2r; PCR3f and PCR3r; PCR4f and PCR4r; and PCR5f and PCR5r (Table A3). Southern hybridization and DNA sequencing were carried out to confirm the RT‐PCR products. As a positive control, pCOK218 DNA was used. As a negative control, PCR reactions with the same primer sets were performed using RNA samples that had not been reverse transcribed.

Total RNA was extracted from both cell cultures using the RNeasy Plant Mini Kit followed by in-column DNase treatment (Qiagen, Hilden, Germany). RNA concentrations were measured using a Nano drop ND-1000 spectrophotometer (Nano drop Technologies, Rockland, DE, USA) and 5 μg of RNA was treated with RQ1 DNase (Promega) according to the manufacturer’s protocols. RNA was precipitated with 2.2 μl of CH₃COONa 3M pH 5.2 and 60.5 μl of ethanol and incubated for 30 min at -20°C. After centrifugation the pellet was washed with 70% ethanol, air-dried, re-suspended in 5 μl of water and used for first-strand cDNA synthesis using the Superscript II Reverse Transcriptase (Thermo Fisher) according to user instructions.