GSM4626297: C2; Oryza sativa Japonica Group; RNA-Seq
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, ThermoFisher), 1 µL RiboLock RNase Inhibitor (EO0381, Thermo Fisher) and 1 µL DNAse I (EN0521, ThermoFIsher). 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 nano chip (Agilent technologies) and the concentraiton was measured with  a Quant-it Ribogreen RNA assay (Life technologies). 1500 ng of RNA was used with the rRNA depletion with Ribo Zero Plant Seed/Root kit (Illumina). Library prep was performed according to manufacturer's instructions. After rRNA depletion, total RNA was purified with RNA clean and concentrator-5 (Zymo Research) and eluted in 10 µL water. Depletion efficiency was checked with Quant-it Ribogreen RNA assay (life technologies). The Truseq stranded total RNA library prep (Illumina) was used for library prep according to manufacturer's instructions. During library prep fragmentation and random priming was performed, followed by first and second strand synthesis, A-tailing and adapter ligation. The cDNA was used for enrichment PCR (13 cycles) and purified with the double AMPure XP cleanup (1:1) (Beckman Coulter). 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. Finally, sequencing was performed on a NextSeq 500 using 2% Phix spike-in (single end reads, 76 bp)
GEO Accession
GSM4626297