Mirnapure mini kit

Total miRNA was extracted using miRNApure Mini Kit (CWBiotech), according to the manufacturer's instruction. Reverse transcription was performed using Taqman microRNA RT Kit (Life Technologies) and Taqman microRNA Assay with specific stem-loop primers (Life Technologies). Real-time PCR was performed using Taqman Universal Master Mix II (Life Technologies). The reactions were incubated at 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min using ABI 7500 real-time PCR system. Results were normalized to the internal control, RNU6B.
Total RNA was extracted using TRIzol reagent (Life Technologies) according to the manufacturer's instruction. Reverse transcription reactions were performed using High Capacity RNA-to-cDNA Kit (Life Technologies). Real-time PCR was performed in ABI 7500 real-time PCR system using SYBR Green PCR Master Mix (Life Technologies). The primers used are shown in Supplementary Table S1. The reactions were incubated at 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. GAPDH was used as internal control.
All reverse transcription reactions included no-template controls, and all PCR reactions were run in triplicates. Relative miRNA or mRNA expression was determined using the comparative C_T (2^−ΔΔCt) method.

sRNAs were isolated from the cambial zone materials of 12 plants in LD3, SD8 and C5 using a miRNApure Mini Kit (CW Biotech, Beijing, China) following the manufacturer’s instructions. Then, the sRNA was polyadenylated by poly (A) polymerase, and first-strand cDNA was obtained from polyadenylated sRNAs using the miRNA cDNA Kit (CW Biotech, Beijing, China) following the manufacturer’s instructions. qRT-PCR was carried out as described: the SYBR Premix Ex Taq™ kit (TaKaRa Bio Inc., Japan) and an ABI 7500 Fast Real-time PCR machine (Applied Biosystems, Foster City, CA, USA) were used to complete the amplification, and the reaction procedure was set up according to the manufacturer’s protocol. Three replicates were performed for each sample with 5.8S rRNA as an internal reference [46 (link)], and we used the 2^-ΔΔCT relative quantification method to calculate relative changes in gene expression [77 (link)]. All the primers are listed in Additional file 3: Table S3.

To analyze SDC1 mRNA expression, total RNA was extracted using TRIzol (Invitrogen) and reverse transcribed into cDNA by using the reverse transcription kit (Takara, Japan). For miRNA isolation and analysis, miRNA was extracted by a miRNApure Mini Kit (cwbio, China), and then, the Bulge-Loop miRNA qRT-PCR Primer Set (RiboBio, China) was utilized to analyze mature miRNA expression. qRT-PCR was performed using SYBR Green Master Mix kit with the StepOne Real-Time PCR System. The relative expression of mRNAs and miRNAs was normalized using GAPDH and U6, respectively, followed by being analyzed by the 2^–ΔΔCt method. U6 and mmu-miR-10a-5p primers were purchased from RiboBio. The primers for SDC1 and GAPDH were designed as follows: SDC1 sense primer, 5′-CCT CAT CTT TGC TGT GTG CC-3′; SDC1 anti-sense primer, 5′-GCT TGG TGG GTT TCT GGT AG-3′; GAPDH sense primer, 5′-CTC AAC TAC ATG GTC TAC ATG TTC-3′; GAPDH anti-sense primer, and 5′-ATT TGA TGT TAG TGG GGT CTC GCT C-3′.