GSM1810036: APP-enriched RNA; Arabidopsis thaliana; miRNA-Seq
Extracts were prepared in 25 mM HEPES (pH 7.5), 100 mM NaCl, 5 mM EGTA, 2 mM MgCl, 0.05% v/v IGEPAL CA-630, 1 mM DTT, 10% Glycerin, 1x Complete Protease Inhibitor (Roche Applied Science, Mannheim) and 50 µM MG-132. Total RNA was extracted using Trizol Reagent. 3 mg of total protein were subjected to Ago-APP. After incubation for 3 h, the affinity-purfied sample was washed and APP-enriched RNA was extracted by addition of Trizol Reagent to the whole reaction. In short, an adenylated adapter was ligated to the 3’ end of the total RNA and APP-enriched RNA by a truncated T4 RNA Ligase 2, followed by the ligation of a 5’ RNA-adapter by T4 RNA Ligase 1 (NEB). After reverse-transcription using a specific primer, the cDNA was amplified by PCR, gel-fractionated and bands or regions corresponding to miRNA inserts were cut out. PCR products from these gel slices were eluted in elution buffer, precipitated and dissolved in water. The quality of the libraries was assessed by qPCR and Bioanalyzer measurements.