RNA-seq of Arabidopsis thaliana Col-0 wildtype and single mutants crwn1, crwn2, crwn3, crwn4 and kaku4 in short-day conditions collected at 150 days after sowing
RNA-seq of Arabidopsis thaliana Col-0 wildtype and single mutants crwn1, crwn2, crwn3, crwn4 and kaku4 in short-day conditions collected at 150 days after sowing
Arabidopsis thaliana Col-0 background plants (wild type, crwn1–4, kaku4) were grown under short-day conditions and sampled at 150 days after sowing. From each rosette, two defined leaf cohorts were dissected: • Basal pool: leaves 1–4 (oldest fully expanded rosette leaves) • Apical pool: leaves 11–14 (most recently formed photosynthetic leaves) Basal and apical pools from the same plant were combined to represent whole-rosette profiles, immediately flash-frozen in liquid nitrogen, and stored at −80 °C. Each biological replicate consisted of pooled tissue from 3–4 plants (one pot), and two biological replicates per genotype were collected. Arabidopsis thaliana Col-0 wild type and nuclear-envelope single mutants (crwn1–4, kaku4) were grown in soil in a controlled-environment growth chamber under short-day photoperiod (8 h light / 16 h dark) at 22 °C with ~60 % relative humidity. Light intensity was maintained at approximately 120–150 µmol m⁻² s⁻¹ of white light. Plants were bottom-watered regularly and fertilized according to standard greenhouse practice. Three pots (3–4 plants per pot) per genotype were established; two/three pots per genotype were used for molecular profiling, and one pot was maintained as a backup under identical conditions. Frozen pooled rosette tissue was ground to a fine powder in liquid nitrogen. Total RNA was extracted using the RNeasy Plant Mini Kit (Qiagen, cat. 74904) following the manufacturer's instructions, including on-column DNase digestion to remove genomic DNA. RNA concentration was measured by Qubit RNA HS assay and purity was assessed by A260/280 and A260/230 ratios. RNA integrity was evaluated on an Agilent Bioanalyzer, and only samples with sufficient yield and high RIN scores were used for RNA-seq library preparation. For each sample, 100 ng of high-quality total RNA was depleted of ribosomal RNA using QIAseq FastSelect reagents according to the manufacturer's protocol. rRNA-depleted RNA was used to prepare strand-specific libraries with the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (NEB, E7760). Briefly, rRNA-depleted RNA was fragmented at 94 °C for 8 min, followed by first-strand cDNA synthesis (25 °C 10 min, 42 °C 50 min, 70 °C 15 min) and dUTP-based second-strand synthesis per kit instructions. After SPRIselect 1.8× cleanup, end repair, A-tailing, adaptor ligation, USER treatment (37 °C, 15 min), and a 0.9× cleanup, libraries were amplified with 12 PCR cycles using NEBNext Ultra II Q5 polymerase. Final libraries were bead-purified, size-checked on a Bioanalyzer (High Sensitivity or DNA 1000), and quantified by Qubit dsDNA HS assay.