title: Cai xin, lettuce and spinach gene expression atlas
design: Cai xin, lettuce and spinach gene expression atlas
library_construction_protocol: On DAG 21, three biological replicates similar in growth were selected. Of these, three to four mature leaves were cut, weighed (minimum total weight 0.5 g) and immediately flash-frozen in liquid nitrogen, and stored at −80℃. For cai xin and lettuce, two to three seeds were placed in each seed holder and covered with a germination dome. The tank was filled with tap water, and the unit was switched on to circulate the water. Light conditions during germination were white light for 24 h (μmol·m-2·s-1), and the temperature was 23-24℃. After 3 days, the radicle appeared in most of the seeds (=DAG 0). On DAG 2, one seedling per seed holder was selected, and those with slower growth removed.  For spinach, seeds were sown on cotton balls, kept in the dark, and sprayed regularly with water to keep seeds damp. Within 3 days, 80% of seedlings germinated (=DAG 0). They were kept in the dark for an additional 2 days until cotyledons were visible. On DAG 2, 8 seedlings were transferred to the seed holders. Light conditions were white light for 24 h (40 μmol·m-2·s-1), and the temperature was 23-24℃. One unit was used per growth condition holding 8 plants, and on DAG 12, five plants were selected to ensure sufficient growth space during the last nine days.  The composition of the half-strength Hoagland's solution was KH2PO4 (0.5 mM), KNO3 (3 mM), Ca(NO3)2 x 4 H2O (2 mM), and MgSO4 x 7 H2O (1 mM). Of the micronutrient stock solution (1000x), 0.5 ml was added to 1L of half-strength Hoagland's Solution. One liter of micronutrients stock solution contained H3BO3 (2.86 g), MnCl2 x 4 H2O (1.81 g), ZnSO4 x 7 H2O (0.22 g), CuSO4 x 5 H2O (0.08 g), Na2MoO4 x 2 H2O (0.025 g), CoCl2 x 6 H2O (0.025 g). One liter of the chelated iron stock solution (200x) contained FeSO4 x 7H2O (5.56 g) and Na2EDTA (7.45 g). 2.5 mL was added to the half-strength Hoagland's solution. KOH was used to adjust the pH to 5.5. Growth medium levels were checked daily and were replenished regularly in each unit to ensure a stable water level. Furthermore, pH and EC (1.3 dSm-1) were monitored every 2-3 days and adjusted by exchanging with fresh growth medium. Plants were harvested on DAG 21. On DAG 5 stress and control conditions were started in half-strength Hoagland's solution. For nutrient stress conditions, the growth medium was modified. For cai xin and lettuce, control conditions were a light intensity of 202.5 μmol·m-2·s-1, an R:B:W ratio of 4:1:1, and a light period of 20h at 25℃. For spinach, the control conditions were a light intensity of 130 μmol·m-2·s-1, an R:B:W ratio of 4:1:1 and a light period of 15h at 22℃. For the light intensity experiment, cai xin and lettuce were grown at 67, 135, 202.5, and 268 μmol·m-2·s-1 at 16h light and 25℃. Spinach was grown at 65, 130, 200, and 260 μmol·m-2·s-1 at 15h light and 22℃. For the day length experiment, cai xin and lettuce were grown at 8h, 12h, 20h, and 24h light at 25℃, 200 μmol·m-2·s-1 and R:B:W 4:1:1. For spinach, the day lengths were 8h, 13h, 18h, 24h light at 22℃, 130 μmol·m-2·s-1 and R:B:W 4:1:1. For the light quality experiments, the R:B:W ratios were 4:1:1, 4:1:0, 3:1:1 and 3:1:0 for all plant species under otherwise control conditions. For the temperature experiments, the temperatures were 20℃, 25℃, 30℃ and 35℃ for all plant species under otherwise control conditions.  In the modified N solution, KNO3 and Ca(NO3)2 were replaced with KCl and CaCI2, respectively, to give the same concentrations of K and Ca as in the original solutions. In the modified P solution, KH2PO4 was replaced by KCl, and in the modified K solution, KH2PO4 and KNO3 were replaced by NaH2PO4 and NaNO3. RNA was extracted using Sigma Spectrum Plant Total RNA kit. Directional mRNA library preparation (poly A enrichment)