GSM2440438: WT-MBP-mRNA-Seq; Arabidopsis thaliana; RNA-Seq
RNA was extracted with TRI Reagent (MRC) Membrane bound polysomes (MBP) were isolated as described (Li et al. 2013). MBP was digested with RNase I (Ambion) and the digestion product was centrifuged in a 15-50% sucrose density gradient. A gradient fractionation system (ISCO) was used to collect the monosome fraction. Monosomal RNA was extracted and resolved on 15% Urea-PAGE. 23-35 nt RNAs were recovered from the gel and phosphorylated by T4 polynucleotide kinase (NEB) and polyadenylated with polyA polymerase (NEB) and reverse transcribed with oligodT linkerM followed by cDNA circularization with CircLigase (Epicenter). rRNAs were removed by hybridization with biotin-conjugated DNA probes followed by capture with Dynabead M-280 Stretavidin (Invitrogen). The remaining cDNA were subjected to library construction following the NEBNext Library Prep kit (NEB).
GEO Accession
GSM2440438