E coli poly a polymerase

Small RNAs from the 48 h time point (uninfected treatment, first biological replicate) were purified from a 15% urea-TBE-polyacrylamide gel as previously described [25 (link)], and selected miRNAs were specifically detected using the Poly(T) Adaptor RT-PCR protocol [30 (link)]. Small RNAs were first poly(A) tailed using the E. coli Poly(A) Polymerase (New England Biolabs, Ipswich, MA, USA) and then reverse transcribed using the SuperScript III First-Strand Synthesis for RT-PCR kit (Thermo Fisher Scientific, Waltham, MA, USA) and a poly(T) adaptor primer [30 (link)] (Table S5). PCR reactions were assembled in 20 μL triplicate reactions using 100 ng cDNA, miRNA-specific forward primer, poly(T) adaptor reverse primer (Table S5), and 2X Brilliant II SYBR Green QPCR Low ROX Master Mix (Agilent Technologies, Santa Clara, CA, USA). Reactions were performed using a Stratagene Mx3000p system (Stratagene, San Diego, CA, USA), and Ct values and dissociation curves were analyzed for specific amplification. Non-template control reactions were also simultaneously set up in triplicate, none of which yielded a Ct value or a dissociation curve (data not shown). PCR products were then analyzed on an agarose gel (4%) to verify the size of the amplimer.

Total RNAs was isolated from the viruliferous and nonviruliferous rice/planthoppers using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA). The poly (A) was added to the 3′ end of sRNAs by E. coli Poly(A) Polymerase (NEB, Ipswich, MA, USA). The first-strand cDNAs of sRNAs were synthesized using Moloney murine leukemia virus (M-MLV) reverse transcriptase (NEB). The vsiRNAs were subjected to reverse transcription PCR (RT-PCR) using the SYBR Green sRNA expression assays. RT-PCR was amplified for 25–28 cycles. U6 snRNA was amplified as endogenous control. The RT-PCR primers are listed in Additional file 18.

For synthesising Cas9 mRNA, the Cas9 gene (including nuclear localisation signal [NLS]) was amplified using Q5 high-fidelity DNA polymerase (NEB) to introduce the T7 promoter upstream of the gene. The quality of the PCR was analysed by agarose gel electrophoresis. The PCR product was purified and used for in vitro transcription using a HiScribe T7 Quick High Yield RNA Synthesis Kit (NEB). RNA was treated with DNase I to remove the DNA template, followed by a spin column purification step (NEB Monarch RNA Cleanup kit). Subsequently, capping (NEB Vaccinia Capping System) and poly A tailing were performed using NEB E. coli poly A polymerase. Finally, the mRNA was purified using Monarch RNA Cleanup kit (NEB) and stored at − 80 °C until use. Cas9 enzyme (EnGen Spy Cas9 NLS) and EnGen Spy Cas9 Nickase were purchased from New England BioLabs GmbH (NEB, Germany).

Expression of piR-24000 within the tissue specimens was analyzed by reverse transcription followed by SYBR-green-chemistry-based quantitative PCR. Universal oligo dT primer (for reverse transcription) and amplification primers (for quantitative PCR) were designed as per Balcells et al. [44 (link)]. For the reverse transcription reaction, 100 ng of total RNA was polyadenylated using E. coli Poly(A) Polymerase (New England Biolabs, Massachusetts, United States), and was subsequently reverse transcribed using the PrimeScript RT Reagent Kit (Takara, Kyoto, Japan) as per the manufacturer’s guidelines. Quantitative estimation of piR-24000 was subsequently carried out by the ABI VIIa PCR system (Applied Biosystems, California, United States) using the TB Green Premix Ex Taq II (Takara, Kyoto, Japan) kit. All experiments were performed in duplicate and results were normalized to the expression of RNU6B and expressed as −∆Ct (negative delta Ct). Primer sequences used are listed in Supplementary Table S2.