Qtower 3g cycler

Total RNA of P. trichocarpa was extracted using the CTAB (cetyltrimethylammonium bromide) method (Jaakola et al., 2001 (link)), and RNA samples were analyzed by agarose gel electrophoresis to ensure integrity. HiScript® II Q Select RT SuperMix for qPCR Kit (Vazyme) was used for reverse transcription, and gDNA Wiper Mix to remove the genome DNA. qRT-PCR was performed using the AceQ® Universal SYBR qPCR Master Mix Kit (Vazyme) following the manufacturer’s instructions. The qRT-PCR was performed on a qTOWER 3G Cycler (Analytik Jena, Germany), and the 2^−ΔΔCT method was used to analyze the relative gene expression level. Primer Premier 5 was used to design the primers, and NCBI primer designing tool (https://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi?LINK_LOC=BlastHome) was employed to check their specificity. The P. trichocarpa actin gene (GenBank ID: XM_002298674) was used as the reference gene (Chu et al., 2014 (link)). All PtrAHL gene-specific primers used for qRT-PCR are listed in Table S1.

The wells of a 96-well polypropylene plate (VWR, white) were filled with 300 µL of PBS alone or supplemented with various concentrations of BSA, Tween 20, and methylcellulose. Some wells were left empty. After 1 h at 37 °C, the wells were washed 3× with PBS and incubated with 50 µL of 10 nM SP6 aptamer for 1 h at 37 °C. The unbound aptamer was washed out with PBS (5×). We used these mild washing conditions without Tween 20 to differentiate the potential distinct effects of the particular blocking agents. Then, 50 µL of qPCR Master Mix with SYBR Green I and aptamer-specific primers were dispensed into the wells. The bound aptamer was quantified by qPCR in a qTower3G cycler (Analytik Jena, Jena, Germany), which also automatically calculated Ct values using a qPCRsoft 4.0 touch software for qTower3G (Analytik Jena). The following cycling conditions were used: 4 min at 95 °C, followed by 45 cycles of 10 s at 95 °C, 7 s at 57 °C, and 14 s at 72 °C.
To check the binding properties of ssDNA and dsDNA, we used the SP6 aptamer and its complementary sequence. For the ssDNA test, 50 µL of 10 nM SP6 aptamer or the complementary sequence to SP6 aptamer were used. For the dsDNA test, 50 µL of a 5 nM or 10 nM mixture of both sense (SP6 aptamer) and anti-sense ssDNA was prepared. All DNA samples were denatured at 95 °C for 5 min and then cooled to RT.

RNA extraction, cDNA synthesis and RT–qPCR were performed as previously described (Liu et al., 2018 ) with a few modifications. Briefly, total RNA was extracted using the Plant RNeasy Extraction Kit (BioTeKe, Beijing, China, Cat. No. RP3302). First‐strand cDNA synthesis was conducted using EasyScript® First‐Strand cDNA Synthesis SuperMix (TransGen Biotech, Beijing, China, Cat. No. AE301‐02). Real‐time RT–PCR (RT–qPCR) was performed using TransStart® Top Green qPCR SuperMix (TransGen Biotech, Beijing, China, Cat. No. AQ131‐04). A qTOWER 3G Cycler and qPCR software (Analytik Jena, Germany) were used to perform the experiment and analyse the data. The 2^−ΔΔCt method was used to determine the relative abundance of transcripts (Livak and Schmittgen 2001). Ubiquitin (UBQ, FG065618) and tubulin (TU, FG067376) were used as internal controls (Lei et al., 2020 ; Liu et al., 2018 ).