Rnase free dnase 1

Trizol^® Reagent (Invitrogen Life Technologies, Paisley, UK) was used to extract total RNA from human spermatozoa following the manufacturer’s instructions. The sample was homogenized in Trizol Reagent (1 mL Trizol^® Reagent/5 × 10⁶ sperm cells) and incubated for 5 min at 20 °C to permit nucleoprotein complexes dissociation. Then, 0.2 mL chloroform/mL Trizol^® Reagent was added and the sample was centrifuged at 12,000× g for 15 min at 4 °C. After centrifugation, the aqueous phase was transferred to a fresh tube and isopropyl alcohol (0.5 mL/mL Trizol^® Reagent) and 1 μL glycogen (20 mg/mL) were added to promote the precipitation of total RNA. After centrifugation at 12,000× g for 10 min at 4 °C, the RNA pellet was washed with 75% ethanol, centrifuged at 7500× g for 10 min at 4 °C and dissolved in an appropriate volume of DEPC treated water. By using a NanoDrop 2000 spectrophotometer (Thermo, Waltham, MA, USA), we assessed the quantity (ng/mL) and purity (260/280 and 260/230 ratios) of total RNAs. Then, the RNA aliquots (10 μg) were treated with 2U DNase I (RNase free DNase I, Ambion, Thermo Fisher Scientific, Massachusetts, USA) according to the manufacturer’s recommendations to remove potential contamination of genomic DNA. The RNAs were then preserved at −80 °C until the next step.

The epidermis of both the prickly and prickleless mutant from 45 days old in vitro grown plantlets was pealed out separately in liquid nitrogen. Epidermis from five plants were pooled to isolate the RNA for one biological replicate. Two such independent replicates for both prickled and prickleless mutant were used for RNA extraction and RNAseq. Total RNA was isolated using Spectrum Plant Total RNA Kit (Sigma-Aldrich, USA) and treated with the RNase free DNaseI (Ambion, Invitrogen) to remove DNA contamination. The quality and quantity of total RNA were analyzed by agarose gel, spectrophotometric analysis (ND1000, NanoDrop Technologies, USA) and Agilent 2100 Bioanalyzer RNA chip (Agilent Technologies Inc., Santa Clara, CA).

The same amount of adipose tissue samples were taken for total RNA isolation. mirVana^TM RNA Isolation Kits (#AM1561, Ambion, USA) were used to isolate total RNA according to the manufacturer's instruction. The isolated total RNA from each sample was preserved at −80°. NanoDrop 2000 spectrophotometer (Thermo Scientific, USA) was used to determine RNA concentration and OD_{260 nm}/OD_{280 nm} absorption ratio, which was controlled in the range of 1.9–2.1. Bioanalyzer 2100 (Agilent, Santa Clara, CA) was used to evaluate the quality of total RNA (RIN>=7 and 28S/18S>=0.7). RNase-free DNase I (Ambion Inc., Texas, USA) was used to eliminate potential genomic DNA contamination.

EV suspension (100 µL) was diluted 3:1 in TRIzol LS (Ambion, Life Technologies, Carlsbad, CA, USA) and held at −80 °C. From EVs, total RNA was extracted, mixed with 10 µL of diethylpyrocarbonate water, and quantified (1 µL) using a BioDrop-μLITE kit (Isogen Life Science, Utrecht, The Netherlands). Them, 100 ng of RNA was treated with RNase-free DNase I (Ambion™ Life Technologies) then reverse transcribed using a HiFlex miScript RT II kit according to the manufacturer’s instructions (Qiagen, Hilden, Germany). Mature miR-29a (#MS00003262), miR-29b (#MS00006566), miR-92 (#MS00006594), miR-155 (#MS00031486) and miR-223 (#MS00003871) were detected by quantitative polymerase chain reaction (qRT-PCR) using an miScript primer assay kit and miScript SYBR Green PCR kit (Qiagen). Amplification of mature microRNA as cDNA was performed in a CFX Connect real-time PCR Detection System (BIO-RAD, Hercules, CA, USA) using 40 cycles of 94 °C for 15 s, 55 °C for 30 s, and 72 °C for 30 s. Reaction specificity was confirmed using the melt curve procedure (65–95 °C, 0.5 °C per 5 s) at the end of the amplification protocol according to the manufacturer’s instructions. A standard curve was used for absolute quantification of microRNA.

Total RNA was isolated from fifth-instar larvae using Trizol Reagent (Invitrogen, Carlsbad, USA) and was treated with RNase-free DNase I (Ambion, Austin, TX, USA), according to the manufacturer’s protocol. cDNA was synthesized with Omniscript reverse transcriptase kit (Qiagen, Hilden, Germany), using manufacturer’s instructions. Putative OfAbd-A and OfUbx genes were identified using the NCBI Blast system. OfAbd-A cDNA fragments were amplified by PCR with the following pair of primers: forward, 5′-ATGGCAGCGGCTGCCCAGTT-3′; and reverse, 5′-TTACGTGGGGACTTTGTTCA-3′. OfUbx cDNA fragments were amplified by PCR with the following pair of primers: forward, 5′-ATGAACTCCTACTTTGAGCAGGGT-3′; and reverse, 5′-TTACGTGGGGACTTTGTTCA -3′. PCR was carried out using KOD -Plus- polymerase (TOYOBO, Osaka, Japan) under the following conditions: 98 °C for 2 min, followed by 30 cycles at 98 °C for 30 s, 55 °C for 30 s, 68 °C for 1 min, and an elongation phase at 68 °C for 10 min. Amplified products were sequenced after cloning into a PJET1.2-T vector (Fermentas, Burlington, ON, Canada). The primers are listed in Table S1.