title: NextSeq 500 sequencing; Effects of root-specific activation of plasma membrane H+-ATPase 1 on genome-wide gene expression in A. thaliana roots
design: Effects of root-specific activation of plasma membrane H+-ATPase 1 on genome-wide gene expression in A. thaliana roots
library_construction_protocol: Roots were harvested, immediately frozen using liquid N2, and stored at −80°C until use. Root samples harvested from two independent grafting experiments were subjected to RNA-Seq analysis. Roots from four plants in each experiment were pooled as one biological replicate. 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 and further cultivated for 1 day under a PPFD of 25 µmol m−2 s−1 (16/8-h light/dark cycle) at 22˚C. The grafted plants were horizontally grown for 7 days under a PPFD of 100–120 µmol m−2 s−1 (16/8-h light/dark cycle) at 22˚C –23˚C in 30 mL of 1/2-Murashige and Skoog (MS) or 1/50-MS salts 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 9.1–10.0 were used for the following preparation. cDNA libraries were constructed using a NEBNext Ultra II RNA Library Prep with Sample Purification Beads (Code E7775; New England Biolabs, Tokyo, Japan), a NEBNext Poly(A) mRNA Magnetic Isolation Module (Code E7490; New England Biolabs), and a NEBNext Multiplex Oligos for Illumina (Index Primers Set 3) (Code E7710; New England Biolabs) according to the manufacturer's instructions.