time-series RNA-seq of xylem
Total RNA was isolated and purified from samples using TRIzol reagent (Thermo Fisher, cat. #15596018) following the manufacturers protocol. RNA quantity and purity were assessed using a NanoDrop ND-1000 spectrophotometer (NanoDrop, Wilmington, DE, USA), and RNA integrity was evaluated with an Agilent Bioanalyzer 2100 (Agilent, CA, USA); only samples with RNA concentration >50 ng/L, RIN >7.0, and total RNA >1 g were used for downstream library preparation. Poly(A)+ mRNA was selectively enriched using oligo(dT) magnetic beads (Dynabeads Oligo(dT), Thermo Fisher, cat. #25-61005) through two rounds of purification. The captured mRNA was fragmented at 94C for 57 min using the NEBNext Magnesium RNA Fragmentation Module (NEB, cat. #E6150S). First-strand cDNA was synthesized using SuperScript II Reverse Transcriptase (Invitrogen, cat. #1896649), followed by second-strand synthesis with E. coli DNA Polymerase I (NEB, cat. #M0209) and RNase H (NEB, cat. #M0297), incorporating dUTP (Thermo Fisher, cat. #R0133) to enable strand specificity. The resulting double-stranded cDNA was end-repaired, A-tailed, and ligated to T-overhang adapters. Library fragments of ~300 bp (50 bp) were size-selected using magnetic beads. The second strand was subsequently degraded by UDG enzyme (NEB, cat. #M0280) prior to limited-cycle PCR amplification (8 cycles: 98C for 15 s, 60C for 15 s, 72C for 30 s; final extension at 72C for 5 min). The final strand-specific RNA-seq libraries were sequenced on an Illumina NovaSeq 6000 platform (LC Bio, Hangzhou, China) in paired-end mode (PE175).