title: GSM9328562: Arabidopsis, ssrp1; Arabidopsis thaliana; OTHER
library_construction_protocol: Nuclear isolation was performed based on a previously published protocol. Approximately 1 g of 12-day-old Arabidopsis thaliana seedlings were ground to a fine powder in liquid nitrogen. The powder was resuspended in ice-cold Honda buffer (0.44 M sucrose, 1.25% (w/v) Ficoll, 2.5% (w/v) dextran T40, 20 mM HEPES-KOH pH 7.4, 10 mM MgCl₂, 0.5% (v/v) Triton X-100, 1 mM DTT, 1× protease inhibitor cocktail, 100 ng/μL tRNA) and washed twice by centrifugation at 3,500 × g for 5 min at 4 °C. Total RNA was subsequently extracted from the isolated nuclei using the RNAprep Pure Plant Plus Kit (DP441; Tiangen). RNA concentration was measured using a NanoDrop spectrophotometer, and samples were stored at -80 °C until further use. FLEP-seq2 libraries were constructed starting from 1 μg of nuclear RNA per sample. First, ribosomal RNA (rRNA) was depleted using pan-plant riboPOOL probes targeting Pan-plant. The rRNA-depleted RNA was then ligated to a 3' linker (S1315S, NEB). The ligated RNA was purified using the RNA Clean & Concentrator-5 kit (R1013, ZYMO). Reverse transcription was performed using a custom cDNA RT primer and a Strand-Switching Primer with Maxima H Minus Reverse Transcriptase (EP0752; Thermo). The thermal cycling conditions were: 42 °C for 90 min, followed by 10 cycles of 50 °C for 2 min and 42 °C for 2 min, with a final enzyme inactivation step at 85 °C for 5 min. The resulting cDNA was amplified by PCR using unique barcode primers from the PCR Barcoding Kit (SQK-PCB109; Oxford Nanopore Technologies). Following amplification, residual primers were removed by treating the library with 1 μL of Exonuclease I (M0293; NEB) at 37 °C for 15 min. The final library was purified using a 0.8× volume of AMPure XP beads (Beckman). Libraries were loaded onto a FLO-MIN106 (R9.4.1) flow cell and sequenced on a MinION Mk1B instrument for 72 hours.