Taq pcr mastermix

We obtained Q5 High-Fidelity DNA polymerase, restriction endonucleases, T4 DNA ligase, and Color Pre-stained Protein Standard from New England Biolabs (USA). Taq PCR Master Mix and DNA ladder were ordered from Tiangen (Beijing, China). Oligonucleotide primers were synthesized by GENEWIZ (Suzhou, China). Plasmids were extracted by E.Z.N.A TM Plasmid Miniprep Kit (Omega Bio-Tek, Inc, USA). Gel extraction and purification were performed by E.Z.N.A TM Gel Extraction Kit (Omega Bio-Tek, Inc, USA) and E.Z.N.A TM Cycle Pure Kit (Omega Bio-Tek, Inc, USA), respectively. We purchased ALA from Adamas Reagent Corporation (China), and SAM was obtained from Xiya Reagent Corporation (China).

Polymerase chain reaction was used to validate the reliability of the high-throughput RNA sequencing data. A Transcriptor First Strand cDNA Synthesis Kit (Roche, Germany) was used for reverse transcription of circRNAs. According to manufacturer’s instructions, appropriate volume of master mix as well as RNA sample were prepared, then the reaction for reverse transcription was initiated at 25°C for 10 min, 55°C for 30 min, and 85°C for 5 min. Then, cDNA and gDNA templates were PCR amplified for 35 cycles using Taq PCR MasterMix (Tiangen, China) following the manufacturer’s protocol, and PCR products were visualized using 2% GelRed-stained agarose gel. To confirm the PCR results, we further performed Sanger sequencing to directly examine the PCR product. To verify the accuracy of the differential expression of circRNAs, qRT-PCR was conducted using a FastStart Universal SYBR Green Master Kit (Roche, Germany). Briefly, the first strand cDNA was synthesized using random hexamer primer and then amplified by SYBR Green Kit following the standard procedure that is denaturation 95°C (10 min) followed by amplification by a total of 40 cycles of 95°C (15 s) and 60°C (1 min) on an ABI7500 system (Applied Biosystems, Foster City, CA, United States). GAPDH was used as an internal control, and PCR primers are listed in Supplementary Table S1.

First-strand cDNA was synthesised from total RNA using genomic DNA (gDNA) and the FastQuant RT Enzyme of the FastQuant RT Kit (with gDNA) (Tiangen, Beijing, China). The reaction mixture (20 μL) contained total RNA (260 ng), 5 × gDNA Buffer (2 μL), 10 × Fast RT Buffer (2 μ), RT Enzyme Mix (1 μL), and FQ-RT Primer Mix (2 μL). The primer mix (25 μL) contained first-strand cDNA (6 μL), 1 μL of each primer, 2 × Taq PCR MasterMix (12.5 μL), and ddH₂O (4.5 μL) (Tiangen). The PCR primers were listed in S1 File. Amplification was carried out under the following conditions: 94°C for 3 min for the initial denaturation of the RNA/cDNA hybrid; 33 cycles at 94°C for 30 s, 55°C for 20 s, and 72°C for 20 s; plus a final extension of 5 min at 72°C. The glyceraldehydes 3-phosphate dehydrogenase cDNA fragment was amplified by intron-spanning primers 5′-tttggcatcgtggaagga-3′ (bases 508–525) and 5′-cgaaggtagaagagtgggagt-3′ (bases 892–872). The PCR product was electrophoresed on a 1% agarose gel and individual bands were visualised by ethidium bromide staining. All experiment was repeated three times.

Total DNA of plants or insects was extracted using the RoomTemp^TM Sample Lysis Kit (Vazyme, P073) according to manufacturer’s protocol. A 412 bp TYLCV fragment was PCR amplified using Taq PCR MasterMix (Tiangen, KT201) and primers V61 and C473 as previously described⁶⁶ . Total RNA of whitefly samples was extracted by TRIzol^TM Reagent (Ambion, 15596018), and reverse transcribed using the FastQuant RT Kit with gDNase (Tiangen, KR106). cDNAs of PEBP4, PEBP4 pep1, PEBP4 pep2, V1 (TYLCV CP), Raf1, and ATG8 were amplified by PrimerSTAR MAX DNA Polymerse (Takara, R045A). RT-qPCR reactions using the PowerUp^TM SYBR Green Master Mix were carried out on the QuantStudio 12 K Flex Real-Time PCR System (ABI) (ABI, A25742). Data were analyzed by the 2^–△△CT relative quantification method, and each biological replicate consisted of three technical replicates.

The total RNA was extracted from cells using the TRIzol reagent (Invitrogen). Cell cycle-related genes were first subjected to reverse transcription-PCR (RT-PCR) using an RT-PCR kit (MBI, Hanover, MD, USA) and then subjected to quantitative real-time RT-PCR using the iQ5 Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA) with SYBR Premix Ex Taq II (TaKaRa). GAPDH mRNA levels were used as an internal normalization control. Fold changes were calculated and normalized using the CT method. PCR was performed with the Taq PCR Master Mix (Tian Gen, Beijing, China).

Cells were collected over the course of phage growth curve experiments for PCR detection of phage DNA integrated into the cyanobacterial genome. Samples were filtered onto polycarbonate 0.22 µm pore-size membrane filters (GE), washed twice with growth medium to remove free phages, and washed once with 3 ml of preservation solution (10 mM Tris, 100 mM EDTA, 0.5 M NaCl, pH 8) [94 (link), 95 (link)]. The filter was flash-frozen in liquid nitrogen and stored at −80 °C. A heat lysis method was used to extract DNA from cells collected on filters [94 (link), 95 (link)]. Filters were resuspended in 10 mM Tris-HCL solution (pH = 8), shaken in a bead-beater (Mini-BeadBeater, Biospec) for 2 min (3450 oscillations/min) without beads. Samples were heated at 95 °C for 15 minutes and the supernatant collected. For detection of integrated phage DNA, 2 µl of intracellular template DNA were used in 20 µl PCRs (Taq PCR MasterMix, Tiangen), as described above. Primers specific for detection of total phage DNA and integration junctions were used (Table S5).