RNA-seq experiment of tissue forming a lateral root after salt treatment in cyp79b2/b3 mutants containing multiple timepoints
RNA-seq experiment of tissue forming a lateral root after salt treatment in cyp79b2/b3 mutants containing multiple timepoints
At 5 day-old (days after transfer to the long day conditions), seedlings were gravistimulated by turning the plates 90 degrees. At 3 hours after turning the mesh including the seedlings was transferred to plates containing 125 mM NaCl or control plates. Samples were taken at 1, 2, 3 and 4 days after treatment. Every  experimental condition includes 3 replicates.  A region of 3 mm around the bending zone was dissected, pooled for ~40 seedlings per sample and frozen in liquid nitrogen. Seedlings that grew away from the plate or stopped growing after transfer were excluded from the analysis.  Seeds were sterilized in 75 % EtOH and 25 % bleach for 5 min, washed twice in 70 % EtOH and once in 96 % EtOH, after which the seeds were left in the laminar flow to dry and were sown using 0.1 % agar on plates containing 0.5 x Murashige-Skoog (MS, Duchefa), 0.1 % M.E.S. Monohydrate (Duchefa) and 1 % Daishin agar (Duchefa) (pH 5.8, with KOH) on top of a mesh with 50 µm openings (Sefar BV). Seeds were stratified at 4 ˚C for 2 days in the dark and grown at a 90˚ angle in a growth chamber under long day conditions (16 h light : 8 h dark, 130 μmol/m2/s LED light) at 21 °C and 70% relative humidity. The samples were ground to powder using a paint shaker. From these samples, RNA was isolated using NZY Total RNA Isolation kit with on-column DNAse treatment (NZYtech). RNA Poly-A enrichment library preparation by Novogene Co., LTD