Rnase free dnase 1 treatment

Total RNAs were extracted from the fourth leaves of the WT and phyB plants as described above. Genomic DNA contamination was eliminated by RNase-free DNase I treatment (Promega, Madison, WI, USA). For miRNA qRT-PCR, miRNA first-strand cDNA was synthesized using the miRcute miRNA First-Strand cDNA Synthesis kit (Tiangen, Beijing, China). The expression levels of 15 random selected miRNAs were quantified by qRT-PCR with the SYBR PrimeScript™ miRNA RT-PCR kit (Tiangen, Beijing, China). For targets qRT-PCR, first-strand cDNA was synthesized using M-MLV reverse transcriptase according to the manufacturer's instructions (Promega, Madison, WI, USA). The expression patterns of selected genes were detected using SYBR Green Real-time PCR Master Mix (PE Applied Biosystems, Foster City, CA, USA) on the Stepone Plus system (Applied Biosystems, USA). The primers used in qRT-PCR are listed in Additional file 1. Three biological replicates were included for each miRNA and target gene in qRT-PCR analysis. The relative expression ratios of each miRNA and target gene were calculated using the delta-delta threshold cycle relative quantification method with the internal control of 5.8s rRNA and OsEF-1α, respectively.

Small RNAs was isolated from the EL and NL tissues using the Plant RNA Isolation Kit (Bioteke, Beijing, China) and purified by RNase-free DNase I treatment (Promega, Madison, USA). Then poly (A) polymerase-mediated RT-qPCR was used to investigate the expression of 21 selected miRNAs. The cDNAs were synthesized from 500 ng of sRNA with the TaKaRa One Step Primescript miRNA cDNA Synthesis Kit (Takara, Dalian, China) following the manufacturer’s recommendations. The 25 μl miRNA reverse transcription reaction mixture was incubated at 37°C for 60 min, followed by 5 s at 85°C. Then SYBR Premix ExTaq II Kit (TaKaRa) was used for the RT-qPCR on a CFX96 Detection System (Bio-Rad, Hercules, California, USA). The reactions were incubated in a 96-well plate at 95°C for 10 s, followed by 45 cycles of 95°C for 5 s and 65°C for 30 s. The melt curve was chosen as the default. The snRNA 18S (GenBank Accession No. D16448.1) was used as an endogenous control. The 2^-ΔΔCT method was used to analysis the relative expression levels. The RT-qPCR was performed using three biological replicates for credible statistical analysis. The miRNA-specific primer sequences and the primers for the reference 18S gene are shown in S4 Table.

Total RNA extractions were performed based on the CTAB method described by Chang et al.^{56 (link)} and adapted to apple^{57 (link)}. Three grams of peel and 9 g of flesh of apple fruit bulk were used for each extraction replicate. Contaminant DNA was removed from the RNA samples using RNase-free DNase I treatment (Promega, Madison, WI, USA) according to the manufacturer’s instructions. RNA integrity was evaluated using 1% agarose gel electrophoresis, whereas the RNA concentration and purity (OD260/OD280 ratio >1.95) were assessed by optical density in a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Montchanin, DE, USA). Three independent RNA extractions were conducted for each sample. First strand cDNA synthesis was performed using the First Strand cDNA Synthesis Kit (Fermentas Life Science, Glen Burnie, MD, USA) following the manufacturer’s instructions.