Rnase free dnase 1

For the expression analyses illustrated in Figures 4A, 7B and 8A, cells were cultivated to an A₆₀₀ of ∼0.5 in a 125 ml culture at 30°C and 15 ml aliquots were removed and subjected to an instantaneous 30 to 39°C upshift for the indicated times. Heat shock induction was terminated through addition of 20 mM sodium azide, and RNA was isolated as above. For induction of YFR057w (Figures 9A, S4), cycloheximide was added to a final concentration of 0.2 mg/ml, 15 ml aliquots were removed at the indicated times and RNA was isolated.
Contaminating genomic DNA was removed from each RNA sample by digestion with RNase-free DNase I (OMEGA Bio-tek Inc #E1091), followed by phenol/chloroform extraction. 0.5 µg purified RNA was used in each cDNA synthesis with ProtoScript RT-PCR Kit (NEB #E6400S). Oligo(dT) primers were used in cDNA synthesis for quantification of Pol II gene transcripts; random primers were used in cDNA synthesis for quantification of SCR1 RNA. 2% of the synthesized cDNA was used in each qPCR, which was performed as described above. Primers were designed to target the 3′UTR of HSP82, YFR057w and PMA1, or the body of SCR1. Their coordinates are: HSP82, +2134 to +2228; YFR057w, +312 to +437; PMA1, +2998 to +3083 and SCR1, +385 to +483. For quantification, SCR1 was used to normalize HSP82 and YFR057w mRNA levels.

Leaves from transgenic and WT seedlings growing to 4 weeks old, incubated under normal conditions, for one day and four days under simulating drought stress with PEG-6000 (10%) or salinity stress with 300 mM NaCl were used for expression analysis. General RNA from different lines was extracted with Plant RNA Kit (Omega Bio-tek, Norcross, GA, United States), which was also purified with RNase-Free DNase I (Omega Bio-tek) and reverse transcribed into cDNA by use of Uscript II (Innovagene biotech, Hunan, China).
The qRT-PCR amplification was performed in 25 µL reaction mixture (innovagene biotech) including 12.5 µL 2 × Taq SYBR Green qPCR Mix, 0.5 µL of 10 µM of each primer, 4 µL 5-fold diluted cDNA, and 7.5 µL of Nuclease-free H₂O, which was performed using the real-time PCR instrument CFX Connect (Bio-Rad, Hercules, CA, USA). Amplification conditions were devised of 94 °C for 3 min, 42 cycles of degeneration at 94 °C for 8 s, annealing/extension at 60 °C for 60 s. The relative expression quantity of the target gene was evaluated based on the method of 2^−ΔΔCT [79 (link)]. Finally, the results were normalized by an internal reference gene AtActin2 for quantitative analysis of relative genes. Each RT-qPCR experiment was reproduced at least three times. The gene-specific amplification primers were listed in Supplementary Table S2.

Genomic DNA of poplar roots was extracted using cetyltrimethylammonium bromide (CTAB). Total RNA was isolated and extracted using a total RNA extraction kit [TRK1001, Lianchuan (LC) Science, Hangzhou, China]. Simultaneously, DNA and ribosomal RNA were removed from total RNA using RNase-free DNase I (E1091, Omega Bio-Tek, Norcross, GA, United States) and ribosomal RNA removal kits (MRZPL116, Illumina, CA, United States), respectively. The RNA was then reverse transcribed using a PrimeScript RT reagent kit (Takara, Dalian, China) according to the manufacturer’s instructions. To confirm that the circRNAs sequenced in poplar roots were actual circRNAs, we used convergent primers (positive control) and divergent primers for PCR (Supplementary Table 1). The PCR products were verified by electrophoresis and sequenced.
To verify the expression of DECs and DE-mRNAs in poplar roots treated with three N forms, RT–qPCR was performed on a Light Cycler^R 480 Real Time PCR System (Roche, United States) using the SYBR Green PCR kit (TaKaRa) as described by Zhou et al. (2020) (link). The relative expression levels were determined by the 2^–ΔΔCt method. The Actin gene was used as an endogenous reference gene for circRNAs and targets. All experiments were repeated three times. Specific primer sequences for the amplification of circRNAs and mRNAs are listed in Supplementary Table 1.