Rnase free water

Total RNA was extracted from the mesocarp samples of four stages (G, R, C, and P), roots, stems, leaves and cotyledon tissues using TRIzol reagent (Invitrogen, United States) according to the manufacturer’s instructions. Total RNA concentration and purity were measured by a NanoDrop 2000 (Thermo Scientific), and total RNA integration and quality were assayed by agarose gel electrophoresis. Then, the genomic DNA was removed by RNase-free DNase I (TaKaRa, Dalian, China), and the total RNA was dissolved in RNase-free water (Tiangen, Beijing, China). The sample for degradome sequencing was composed of equal amounts of the four stage samples to obtain a sufficiently large sample.

Rabbit primary chondrocytes (iCell Bioscience Inc., Shanghai, China); Fetal Bovine Serum (Gibco, 10099141); Trypsin 0.25% EDTA (Gibco, 25200114); DMEM High Glucose (HyClone, SH30022.01); Penicillin–Streptomycin Solution (HyClone, SV30010); DPBS (Gibco, C14190500BT); Recombinant Human IL-1β (Invitrogen, USA); Cell Counting Kit-8 (Keygen, Nanjing, China); Annexin V-FITC/PI Apoptosis Detection Kit (Keygen, Nanjing, China); Diacerein (Aladdin, Shanghai, China); dimethyl sulfoxide (Sigma-Aldrich, Germany); RNase-free water (Tiangen, Beijing, China); Revertra Ace (TOYOBO, Japan); Recombinant Rnasin Ribonuclcase Inhibitor (Promega, USA); Advanced SYBR Green Supermix (Bio-Rad, USA); Oligo (dT) 18 Primer (TaKara, Japan); TRI Reagent (MRC, USA); HPLC-grade MeOH (Fisher Scientific, NJ, USA). Analytical-grade solvents for extraction and chromatography (Beijing Beihua Fine Chemicals Company, Beijing, China); ODS-A C18 reversed-phase silica gel (50 μm, YMC).

Synthesized cDNA libraries were diluted with RNase-free water (Tiangen, Beijing, China) at a ratio of 1:3 before qRT-PCR. The qRT-PCR reaction system was 10 µL, including 5 µL of SYBR Green Premix Ex Taq (TaKaRa), 3µL of RNasefree H₂O (Tiangen), 0.5 µL of forward primer, 0.5 µL of reverse primer, and 1 µL of cDNA. In addition, qRT-PCR reaction conditions were as follows, 95℃ for 3 min, followed by 95℃ for 10 s, the primer-specific annealing temperature for 20 s, and 72℃ for 20 s; these were then repeated 40 times. GAPDH, β-actin, and U6 (for miRNA) were used as the internal control. All relative gene expression levels were collected with CPX Connect Real-Time System (Bio-Rad, Hercules, CA). The 2^−ΔΔCt method was used to calculate qRT-PCR data. Each sample was assayed in triplicates.