title: GSM4017134: IS-10dai Replicate 2 [L]; Beta vulgaris; RNA-Seq
library_construction_protocol: Whole root tissue of infected and control plants was collected at 4 and 10 dai, were then washed in water, frozen in liquid nitrogen and stored at -80 °C until RNA extraction. For each time point two independent biological replicates were collected and each replicate consisted of a pool of six individual plants. Total RNA was extracted from whole roots of infected and uninfected plants at 4 and 10 dai with the RNeasy Plant Mini Kit (Qiagen) according to the manufacturer's protocol, with an additional sonication step for 30 seconds after addition of buffer RLT, and were then stored at -80 °C. The quantity and quality of each RNA sample was controlled by a NanoDrop 2000c (Thermo Fisher Scientific, Belgium). For each sample, 2 µg of total RNA was used for library preparation using QuantSeq 3' mRNA-Seq Library Prep Kit (Lexogen) as following: An oligodT primer containing an Illumina-compatible-linker sequence at its 5´ end was hybridized to the RNA and reverse transcription was performed. After first strand synthesis the RNA was removed and second strand synthesis was initiated by random priming and a DNA polymerase. The random primer also contained an Illumina-compatible-linker sequence at its 5´ end. The second strand synthesis was followed by a magnetic bead-based purification step. Amplification of libraries was performed by addition of external barcodes during the PCR amplification step. In the final step the double-stranded libraries were purified by magnetic beads to remove all reaction components. The libraries were checked using an Agilent Bioanalyzer 2100 (Agilent). After cluster generation, 16 libraries were sequenced on a NextSeq 500 Illumina sequencing platform to produce single-end 76 bp reads. The samples were multiplexed to minimize lane effects. Library construction and sequencing were carried out by NXTGNT, the UGent sequencing reference center (Faculty of Pharmaceutical Sciences, Ghent University, Belgium).