title: Illumina HiSeq 2000 sequencing; RNA-seq analysis of Arabidopsis thaliana cells treated with RN1 and 2,4-D
design: RNA-seq analysis of Arabidopsis thaliana cells treated with RN1 and 2,4-D
library_construction_protocol: Samples were treated with 50 µM RubNeddin1 (RN1, Chembridge ID 6389186), 2,4-D (CHEBI:28854), or an equal volume of DMSO.  PSB-D cell cultures were grown in continuous darkness in MSMO medium. Cultures were maintained in MSMO medium (4.43 g/L MSMO, Sigma-Aldrich), 30 g/L sucrose, 0.5 mg/L NAA, 0.05 mg/L kinetin, pH 5.7 adjusted with 1 M KOH) at 25°C in the dark, 130rpm. Every 7 days the cells were subcultured (1/10) in fresh medium. Total RNA was extracted from each 10 mL cell culture sample, for each of at least 3 biological replicates, with the RNeasy Plant Kit (Qiagen) according to the manufacturer's instructions. RQ1 RNase-free DNase (Promega) was used for the on-column DNase digestion step.   Preparation of cDNA libraries: Purification of poly-A containing mRNA molecules; mRNA fragmentation; Random primed cDNA synthesis (strand specific); Adapter ligation and adapter specific PCR amplification.