title: Illumina HiSeq 2500 paired end sequencing; RNA sequencing of rice root in salt stress condition (250mM NaCl treatment)
design: RNA sequencing of rice root in salt stress condition (250mM NaCl treatment)
library_construction_protocol: After sufficient growth, plants were divided into two batches, where one batch was treated with 250 mM NaCl and other batch consists of control, which was remained in soil trays placed in water. Salt-treated samples were washed with 250 mM NaCl prior to sample collection. Seedlings and root morphology were analyzed, and sample collection was done for each batch from four independent physiological experiments, which was carried out at an interval of 2 weeks. Seeds of japonica rice cv. Chilbo were sterilized with sodium hypochlorite and germinated in MSO media in an incubator (14-h light/10-h dark, 28 °C (day)/22 °C (night), humidity 80%; Yonghwa Science, Daegu, Korea). After 1 week of germination in the incubator, seedlings were transferred and grown in plastic tray containing commercial soil supplemented with water for a period of 4 weeks under growth chamber condition.  Total RNA was isolated using RNAiso kits (Takara Bio, Shiga, Japan) and first-strand cDNA was synthesized with MMLV Reverse Transcriptase (Promega, WI, USA) and the oligo(dT) 15 primer. Synthesized cDNAs were amplified using a SYBR Premix Ex Taq (Takara) before qRT-PCR was performed on a Rotor-Gene Q instrument system (Qiagen, Hiden, Germany). "The sequencing library is prepared by random fragmentation of the DNA or cDNA sample, followed by 5' and 3' adapter ligation. Alternatively, ""tagmentation"" combines the fragmentation and ligation reactions into a single step that greatly increases the efficiency of the library preparation process. Adapter-ligated fragments are then PCR amplified and gel purified."