GSM5225006: Leaf_Mock_1; Arabidopsis thaliana; ncRNA-Seq
Tissue was ground in liquid nitrogen and small RNA was isolated using the Zymo Direct-zol kit RNA was incubated in 0.1M Tris pH 9.0 for 45 min at 37°C for deacylation. Demethylation was perfomed as described (Zheng et al., 2015): RNA was incubated at room temperature for 2 hours with 160 pmol of wt AlkB and 200 pmol of D135S AlkB in abuffer containing 300 nM KCl, 2mM MgCl2, 50mM (NH4)2Fe(SO4)2×6H2O, 300mM 2-ketoglutarate, 2mM L-ascorbic acid, 50mg/mL nuclease free BSA, and 50 mM MES pH 5.0. RNA was then separated on a 10% TBE-Urea gel (Novex), the region of each lane corresponding to 55 – 100 nt was isolated and RNA was recovered by passive elution into 300mM sodium acetate pH 5.2 followed by ethanol precipitation. Libraries for Illumina® sequencing were prepared using the NEBNextÒ Multiplex Small RNA Library Prep Set for Illumina® (Set1) according to the manufacturer's instructions.
GEO Accession
GSM5225006