GSM5026118: GROseq_wt; Arabidopsis thaliana; OTHER
Approximately 5 million nuclei were run-on in 3×NRO-reaction buffer with 20 nM limiting cytidine triphosphate (CTP) at RT as described (PMID 27729530). Reactions were stopped by addition of 750 µl TRIzol LS (Fisher Scientific), and RNA was purified according to the manufacturer's manual.  RNA pellets were resuspended in 18 µl ddH2O + 0.05% Tween (dH2O+T), 2 µl 10x fragmentation buffer [100 mM ZnCl2, 10 mMTris-HCl (pH 7.5)] added and incubated at 70°C for 15 minutes. Fragmentation was stopped by addition of 2.5 µl of 100 mM EDTA and 500 µl of ice cold GRO binding buffer [0.25× saline-sodium-phosphate-EDTA buffer (SSPE), 0.05% (vol/vol) Tween, 37.5 mM NaCl, 1 mM EDTA]. BrdU enrichment was performed as described (PMID 29779944). For RNA end-repair and decapping by the pyrophosphohydrolase RppH (NEB), RNA pellets were dissolved in 8 µl TET [10 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.05% Tween20] by vigorous vortexing, heated to 70°C for 2 minutes and quick chilled on ice. After a quick spin, 22 µl Repair MM [3 µl 10x PNK buffer, 15.5 µl dH2O+T, 0.5 µl, SUPERase-In RNase Inhibitor (10 U), 2 µl PNK (20U), 1 µl RppH (5U)] was added, mixed by flicking and incubated at 37°C for 1 hour. To ensure phosphorylated 5'ends, 0.5 µl 100 mM ATP was subsequently added and the reactions incubated for another 45 minutes at 37°C. The IP was performed two times. sRNA libraries were prepared using the NEB sRNA kit (E7330) as described by the manufacturer. Libraries were amplified for 15 cycles, and size selected on an 8% PAGE/TBE gels to 150–250 base pairs. DNA eluted from gel slices was precipitated with ethanol and resuspended in Tris buffer (Qiagen) before sequencing.