Silamat s5 vibrator

Approximately 30 mg of plant tissue was placed in a 1.5 mL Eppendorf LoBind tube containing glass beads 1.7–2.1 mm diameter (Carl Roth, Karlsruhe, Germany) and 100 µL of TRI-Reagent (Zymo Research, Irvine, US). The Eppendorf tube was firmly attached to a SILAMAT S5 vibrator (Ivoclar Vivadent, Schaan, Liechtenstein) to disrupt and homogenize the tissue for 2 × 15 s. Total RNA was extracted using Direct-zol™ RNA MiniPrep System (Zymo Research, Irvine, CA, USA) according to the manufacturer’s protocol. The RNA Integrity Numbers and RNA concentration were determined by RNA ScreenTape system with 2200 Tapestation (Agilent Technologies, Santa Clara, CA, USA) and RNA HS Assay Kit with Qubit 3.0 Fluorometer (Thermo Fisher Scientific, Waltham, MA, USA), respectively.

Left ventricular samples (4‐5/group) were placed in a 1.5 mL Eppendorf LoBind tube containing glass beads (1.7‐2.1 mm diameter, Carl Roth) and 500 µL of VRX buffer (Viogene Biotek). The Eppendorf tube was firmly attached to a SILAMAT S5 vibrator (Ivoclar Vivadent) to homogenize the tissues. Total RNA was isolated using Viogene miTotal RNA Extraction Miniprep System (Viogene Biotek) according to the manufacturer's protocol. RNA concentration was measured with RNA HS Assay Kit with Qubit 3.0 Fluorometer (Thermo Fisher Scientific). RNA Integrity Number (RIN) was determined using RNA ScreenTape system with 2200 Tapestation (Agilent Technologies).
NEBNext Multiplex Small RNA Library Prep Set for Illumina (New England Biolabs) was used for small RNA library construction according to the manufacturer's protocol. Libraries were quantified and qualified using High Sensitivity DNA1000 ScreenTape system with 2200 Tapestation (Agilent Technologies) and dsDNA HS Assay Kit with Qubit 3.0 Fluorometer (Thermo Fisher Scientific). Libraries were pooled and diluted to 1.8 pM for 2 × 43 bp paired‐end sequencing on the NextSeq 550 Sequencing System (Illumina) at the Xenovea Ltd. using 75‐cycle High Output v2 Kit according to the manufacturer's protocol.