RNA-Seq of Arabidopsis thaliana: Monopotassium phosphate (KH2PO4) or Conventional Fertilizer (CF), 8 ppm, biological replicate 2
Total RNA from Arabidopsis seedlings were isolated with TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions with some modifications. To improve the performance and purity, 1/10 (v/v) of 7.5 M LiCl was added before the ethanol-precipitation step and a washing step with chloroform: isoamyl alcohol (241, v/v) was also included. The purified RNA was dissolved in 50 L of RNase-free water (Ambion, Austin, TX, USA). Each of the two performed libraries, which were sequenced per analyzed condition (biological replicates), was generated by combining a set of 20 seedlings. RNA integrity was evaluated by horizontal electrophoresis with 1% agarose gels and chip-based capillary gel electrophoresis using a Bioanalyzer system (Agilent Technologies, Santa Clara, CA, USA). RNA concentration was determined using a visible /UV/ BioSpecnano (Shimadzu) spectrophotometer. A 500ng of RNA sample was used as input material for the cDNA library preparation. Using the TruSeq RNA Sample Preparation Kit (Illumina, San Diego, CA, USA) and index codes to identify each sample independently.