title: GSM9363983: non4N_Arabi-flower-rep1; Arabidopsis thaliana; ncRNA-Seq
library_construction_protocol: 1 ml of Trizol reagent (Invitrogen) was added to lyse the cells completely. Subsequently, 200 µl of trichloromethane was added and mixed thoroughly. The solution was then centrifuged at 12,000 rpm for 15 min at 4°C. The upper aqueous phase (450 µl) was transferred into a new 1.5 ml EP tube, followed by the addition of 900 µl of absolute ethanol. The solution was mixed by inversion, left to stand at -30°C for 30 min, and then centrifuged again at 12,000 rpm for 15 min at 4°C, until a distinct precipitate formed at the bottom of the tube. After removing the supernatant, the pellet was washed twice with 80% ethanol, air-dried for 2-3 min at room temperature, and dissolved in DEPC-treated water. RNA concentration was measured using a Nanodrop spectrophotometer, and the sample was diluted to a concentration of 100 ng/µl. 100 ng RNA with 0.05 µl of 20 nM EM or RM spike-in control oligos was ligated to a 0.55 µM 3' randomized adapter using 25 U/µl T4 RNA ligase 2 truncated KQ (NEB) in 0.83X T4 RNA ligase reaction buffer (NEB), supplemented with 20% PEG 8000 (NEB) at 25°C overnight. The ligated RNA was then annealed with 1.28 µM RTP. To remove the 3' adapter-RTP byproduct, 3.33 U/µl 5' deadenylase (NEB) and 0.2 U/µl lambda exonuclease (NEB) were added, followed by incubation at 30°C for 1 h and 37°C for 2 h, respectively. Subsequently, the product was ligated to a 5' randomized adapter using 0.4 U/µl of T4 RNA ligase 1 (NEB) in 0.74X T4 RNA ligase reaction buffer, supplemented with 0.7 mM ATP and 17.3% PEG 8000 (NEB) at 25°C for 1 h. Reverse transcription was carried out using 5 U/µl of ProtoScriptII reverse transcriptase (NEB) in 1X ProtoScriptII RT reaction buffer (NEB), containing 0.5 mM dNTPs (Promega) and 10 mM DTT (NEB), at 50°C for 1 h. The resulting cDNA was then amplified using 0.02 U/µl of KOD-Plus-Neo (TOYOBO) in 1X PCR Buffer for KOD-Plus-Neo (TOYOBO), along with 0.5 µM primers, 1.5 mM MgSO4 (TOYOBO), and 0.2 mM dNTPs (TOYOBO). After PCR amplification, the cDNA was gel-purified on a 6% polyacrylamide gel to remove adapter dimers and sequenced using the Illumina NovaSeq platform.