title: GSM9104465: mRNA-rep2; Arabidopsis thaliana; RNA-Seq
library_construction_protocol: Total RNA from all species except yeast was isolated using the TransZol Up reagent (TransGen Biotech) following manufacturer's instructions. For bacterial RNA extraction, pellets obtained from 300 mL of culture were first pretreated. The bacterial pellet was thawed on ice and resuspended in 6 mL of 2 mg/mL lysozyme solution, then incubated at 37 °C for 5 min to initiate cell wall digestion, followed by cooling on ice for 30 min. Subsequent RNA extraction was performed using the TransZol Up reagent. Arabidopsis and mouse tissues were flash-frozen in liquid nitrogen and homogenized using a pre-chilled mortar and pestle, while monolayer-adherent human cells were washed with 1×PBS and subsequently lysed using TransZol Up. Yeast total RNA extraction employed a phenol-based lysis method. Yeast cell pellets were resuspended in 12 mL of TES buffer (10 mM Tris-HCl pH 7.5, 10 mM EDTA, 0.5% SDS), followed by immediate mixing with 12 mL of phenol through vigorous vortexing for 30 seconds. The mixture was incubated at 65 °C for 1 h to facilitate cell lysis. After incubation, the samples were centrifuged at 12,000 g for 5 minutes at 4 °C, and the aqueous phase was carefully collected. The remaining purification steps were carried out following the standard TransZol Up protocol, following manufacturer's instructions. Following total RNA isolation, all samples were treated with 1 unit/μg of DNase I (Roche) at 37 °C for 1 h to remove genomic DNA contamination. RNA was then purified by phenol/chloroform extraction and ethanol precipitation. For all species except bacteria, poly(A) RNAs were isolated from total RNA using Oligo d(T)₂₅ Magnetic Beads (New England Biolabs) according to manufacturer's guidelines. Subsequently, 20 units RNase H (New England Biolabs) was added, and incubation continued at 37 °C for 40 min. To isolate the cDNA, 30 μL of 2 × RNA loading dye was added to the sample, which was then resolved in a 7 M urea/1 × TBE/10% polyacrylamide gel. The gel was stained with SYBR Gold nucleic acid gel stain (Invitrogen) and visualized under UV transillumination. Gel fragments corresponding to 150–-450 nt cDNA were excised and broken to pieces in 1.5 mL tubes using RNase-free pipette tips. The gel pieces were mixed with 600 μL 0.4 M NaCl and incubated overnight at 4 °C to elute the cDNA. The cDNA was then ethanol-precipitated and resuspended in RNase-free water. PCR amplification was performed using 11.25 μL of the cDNA as a template in a 25 μL total reaction volume. The library was then constructed using the NEBNext® Multiplex Small RNA Library Prep Set, following the manufacturer's recommended cycling conditions (15 cycles). The amplified products were mixed with 6 × DNA loading dye, then resolved in a 1 × TBE/10 % polyacrylamide gel, stained with SYBR Gold, and visualized under UV transillumination. Library fragments spanning 150–-450 bp were excised, gel-extracted, and resuspended in 15 μL of TE buffer. The libraries were then sequenced on an Illumina NovaSeq 4000 system (paired-end, 150 bp) at Berry Genomics (Beijing).Subsequently, 20 units RNase H (New England Biolabs) was added, and incubation continued at 37 °C for 40 min. To isolate the cDNA, 30 μL of 2 × RNA loading dye was added to the sample, which was then resolved in a 7 M urea/1 × TBE/10% polyacrylamide gel. The gel was stained with SYBR Gold nucleic acid gel stain (Invitrogen) and visualized under UV transillumination. Gel fragments corresponding to 150–-450 nt cDNA were excised and broken to pieces in 1.5 mL tubes using RNase-free pipette tips. The gel pieces were mixed with 600 μL 0.4 M NaCl and incubated overnight at 4 °C to elute the cDNA. The cDNA was then ethanol-precipitated and resuspended in RNase-free water. PCR amplification was performed using 11.25 μL of the cDNA as a template in a 25 μL total reaction volume. The library was then constructed using the NEBNext® Multiplex Small RNA Library Prep Set, following the manufacturer's recommended cycling conditions (15 cycles). The amplified products were mixed with 6 × DNA loading dye, then resolved in a 1 × TBE/10 % polyacrylamide gel, stained with SYBR Gold, and visualized under UV transillumination. Library fragments spanning 150–-450 bp were excised, gel-extracted, and resuspended in 15 μL of TE buffer. The libraries were then sequenced on an Illumina NovaSeq 4000 system (paired-end, 150 bp) at Berry Genomics (Beijing).