Lightcycle96 machine

Total RNA was extracted from the Total RNA Isolation Reagent (Biosharp, Cat# BS259A). Reverse transcription was performed with FastKing kit cDNA (Tiangen, Cat# KR118-02). Quantitative PCR was performed with Powerup SYBR Master Mix (Applied Biosystems, Cat# A2577) using a Roche Light Cycle96 machine. All these procedures were performed according to the manufacturer’s protocols. Gapdh was used as a normalized control. All RT-qPCR primer sequences were provided in the Table S1.

Total RNA was extracted from leaf tissue using the TRIzol reagent (Invitrogen) according to the manufacture’s protocol. sRNA northern blot analysis was performed as previously described [41 ]. For nta-miR6019 northern blot, we used the locked nucleic acid (LNA) modified oligonucleotide probe as previously described [48 (link)]. Probe sequences are listed in S3 Table. Quantitative real-time PCR was carried out using SYBR Green fluorescence and a Light Cycle 96 machine (Roche). The threshold cycle (Ct) value was automatically calculated by the Roche Light Cycle 96 1.1 system software and the ΔΔCt method was used to calculate the relative expression levels [49 (link)]. GAPDH was used to normalize the expression of genes in various RNA samples. Three independent biological replicates and three technical replicates of each sample were used for quantitative PCR analysis. Primers used in the experiments are listed in S3 Table.

Leaf samples were collected as described previously (Yan et al., 2017 (link)). Leaf1 to leaf5 (Figure 5A) were collected for gene expression analysis. Samples were picked and immediately frozen in liquid nitrogen and stored at –80°C. The total RNA was extracted by RNAprep pure Plant Kit according to the manufacturer (Tiangen, China). 1.0 µg of RNA was used to synthesize the cDNA using PrimeScript RT Master Mix Kit (TaKaRa, China). qRT-PCR assays were performed by using SuperReal PreMix SYBR Green Kit (Tiangen, China) on the LightCycle96 machine (Roche, Switzerland) as reported before (Shen et al., 2016 (link)). The relative expression levels of genes were compared with the expression of A. annua β-actin and calculated by the 2^-ΔCt. Three biological×four technical replicates were measured for each sample. All the primers used in qRT-PCR were listed in Supplemental Table 2.