Effects of root-specific deficiency of each cytokinin receptor AHK2, AHK3, or AHK4 on genome-wide gene expression in shoots and roots of Arabidopsis thaliana
Effects of root-specific deficiency of each cytokinin receptor AHK2, AHK3, or AHK4 on genome-wide gene expression in shoots and roots of Arabidopsis thaliana
Shoots and roots were harvested, immediately frozen in liquid nitrogen, and stored at - 80 °C until use. Sampling was performed independently three times, with three grafted plants pooled as one biological replicate for each sample. Seeds were placed in the dark at 4˚C for 3 days to break dormancy. Seedlings were grown vertically under a PPFD of 25 µmol m⁻² s⁻¹ (16/8-h light/dark cycle) at 22˚C. Micrografting was performed aseptically using 5-day-old seedlings, which were cut perpendicularly in the hypocotyl with an injection needle tip. The obtained scion was connected with the partner rootstock in a silicon microtube. The grafted plants were incubated for 5 days under a PPDF of 50 µmol m⁻² s⁻¹ (constant light) at 27˚C. The grafted plants were horizontally grown for 7 days under a PPFD of 70 µmol m⁻² s⁻¹ (16/8-h light/dark cycle) at 22˚C in 30 mL of 1/2-Murashige and Skoog (MS) without vitamins supplemented with 0.05% (w/v) MES-KOH (pH 5.7), 1% (w/v) sucrose, and 0.8% (w/v) purified agar. Frozen samples were ground with a Multi-Beads Shocker (Yasui Kikai, Osaka, Japan) using zirconia beads (diameter: 5 mm). Total RNA was extracted using an RNeasy Plant Mini Kit (Qiagen, Tokyo, Japan) according to the manufacturer's instructions with on-column DNase digestion (Qiagen). RNA quality was evaluated using a Qubit RNA IQ Assay Kit with a Qubit 4 Fluorometer (Thermo Fisher Scientific, Tokyo, Japan). The RNA samples for which RNA IQs were 9.5–10.0 were used for the following preparation. cDNA libraries were constructed using a NEBNext Ultra II Directional RNA Library Prep (E7760S, New England Biolabs, Ipswich, MA, USA), SPRIselect (B23317, Beckman Coulter), a NEBNext Poly(A) mRNA Magnetic Isolation Module (E7490S, New England Biolabs), and a NEBNext Multiplex Oligos for Illumina (E7710S, New England Biolabs) according to the manufacturer's instructions.