GSM4727532: G2; Oryza sativa Japonica Group; miRNA-Seq
Root tips and galls were collected from control and inoculated plants respectively, three days post inoculation (3 dpi). Plant materials were immediately frozen in liquid nitrogen and stored at -80 °C. Three biological replicates of control and treated plants were made. Each biological replicate consisted of ca 9 plants. Frozen samples were ground and total RNA was extracted using the ZR Plant RNA Miniprep kit (Zymo Research) according to the manufacturer's instructions. The bead beater step was performed using a speed of 4 m/s for 45 s. All centrifugal steps were performed at 16 000 x g.  For DNase treatment 1 µg of total RNA per sample was combined with 3.6 µL DNase I buffer + MgCl2 (B43, Thermo Fisher), 1 µL RiboLock RNase Inhibitor (EO0381, Thermo Fisher) and 1 µL DNAse I (EN0521, Thermo Fisher). Afterwards RNase-free water was added until a total volume of 36 µL was reached followed by incubation for 30 minutes at 37 °C. Finally, 4 µL 25 mM EDTA was added before incubating for 10 minutes at 65 °C. The quality of total RNA of gall and control samples was verified with a RNA 6000 pico chip (Agilent technologies) and the concentration was measured with a Quant-it Ribogreen RNA assay (Life technologies). 350 ng of RNA was used with the Small RNA seq library prep for Illumina (Lexogen) with the use of a small RNA dual indexing kit (Lexogen). Library prep was performed according to manufacturer's instructions. Briefly, 3' adapters were ligated and excess 3' adapters were removed. 5' adapters were then ligated, followed by cDNA synthesis of the ligated RNA. The cDNA was used for enrichment PCR (14 cycles) and purified with  AMPure XP purification (1:1.8) (Beckman Coulter). Adapter dimers were removed using a 8 % native PAGE gel (Life Technologies). The quality was checked with a High sensitivity DNA chip (Agilent technologies). Quantification of the libraries was done with a qPCR assay according to Illumina protocol to enable equimolar pooling of libraries. An additional purification step was performed on E-gel EX agarose gel (2 % agarose). Finally, sequencing was performed on a Hiseq 3000 using 5 % Phix spike-in (single end reads, 50 bp).