design: Transcriptome analysis of Arabidopsis shoots and roots whose nitrate levels and IPT3 expression are manipulated in a shoot-specific manner
library_construction_protocol: Shoots and roots were harvested, immediately frozen using liquid N2, and stored at −80°C until use. Sampling was done three times independently; each of 9, 6, and 7 grafted plants was pooled as one biological replicate.  Culture media Three different compositions of solid media were used for in vitro culturing of plants as follows: (i) 2.5 mM NH4+ medium; half-strength Murashige and Skoog (1/2MS) modified basal salt mixture without nitrogen (M531; PhytoTech Labs, Lenexa, KS, USA) supplemented with 1.25 mM (NH4)2SO4, 10.0 g L-1 sucrose, 1.0 g L-1 MES, and 4.0 g L-1 gellan gum (Fujifilm Wako, Osaka, Japan) (adjusted to pH 6.7 with KOH). (ii) 10 mM NO3- and 2.5 mM NH4+ medium; 1/2MS modified basal salt mixture without nitrogen (M531; PhytoTech Labs) supplemented with 10 mM KNO3, 1.25 mM (NH4)2SO4, 10.0 g L-1 sucrose, 1.0 g L-1 MES, and 4.0 g L-1 gellan gum (Fujifilm Wako) (adjusted to pH 6.7 with KOH). (iii) No N medium; 1/2MS modified basal salt mixture without nitrogen (M531; PhytoTech Labs) supplemented with 2.0 g L-1 sucrose, 0.5 g L-1 MES, and 5.0 g L-1 gellan gum (Fujifilm Wako) (adjusted to pH 5.7 with KOH).  Transfer experiments using grafted plants  The surface-sterilized seeds were placed in the dark at 4°C for 3 days to break seed dormancy. Then, the seeds (ca. 120 seeds per dish) were sown in rectangle dishes (length, 140 mm; width, 100 mm; depth, 20 mm; Eiken Chemical Co. Ltd., Tokyo, Japan) containing 50 ml of the 2.5 mM NH4+ medium and grown vertically at 22°C for 4 days under a photosynthetic photon flux density (PPFD) of 25 µmol m-2 s-1 (16/8-h light/dark cycle). Micrografting using the 4-day-old seedlings was performed aseptically in rectangle dishes containing 50 ml of the 2.5 mM NH4+ medium. The seedlings were cut perpendicularly in the upper hypocotyl with an injection needle tip (NN-2613S; TERUMO, Tokyo, Japan) on membrane filter (HAWP04700; Merck Millipore, Darmstadt, Germany). The obtained scion was connected with the partner rootstock through a 0.4 mm silicon microtube (1-8194-04; AS ONE, Osaka, Japan). The grafted plants were incubated in a growth chamber at 27°C for 5 days under a PPFD of 50 µmol m-2 s-1 (constant light) and further cultivated at 22°C for 24 hours under a PPFD of 25 µmol m-2 s-1 (16/8-h light/dark cycle). Successfully grafted plants without adventitious root formation were selected for size uniformity. The grafted plants were transferred to round dishes (diameter, 90 mm; depth, 20 mm; AS ONE) containing 30 ml of the 10 mM NO3- and 2.5 mM NH4+ medium and grown for 24 hours under a 25 µmol m-2 s-1 (constant light). Finally, the plants were transferred to the round dishes containing 30 ml of the no N medium and grown for 5 days under a 25 µmol m-2 s-1 (constant light). It should be noted that surfaces of the no N medium were covered with cellophane sheets to allow nondestructive sampling of the roots. Frozen samples were ground with a Multi-Beads Shocker (Yasui Kikai) using zirconia beads (diameter: 5 mm). Total RNA was extracted using an RNeasy Plant Mini Kit (Qiagen) 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 (ThermoFisher Scientific). The RNA samples for which RNA IQs were 9.4–10.0 were used for the following preparation. cDNA libraries were constructed using a NEBNext Ultra II RNA Library Prep with Sample Purification Beads (E7775S, New England Biolabs, Tokyo, Japan), 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.