title: Illumina HiSeq 4000 paired end sequencing; Effects of arbuscular mycorrhiza on transcriptome of fruits from a commercially used tomato cultivar grown in hydroponics
design: Effects of arbuscular mycorrhiza on transcriptome of fruits from a commercially used tomato cultivar grown in hydroponics
library_construction_protocol: Fruits of eight mycorrhizal and non-mycorrhizal plants each were harvested, seeds removed, transferred to liquid nitrogen and stored at -80 C. Samples were lyophilized, homogenized and pooled to unify two fruits in one sample.  Plants were grown in perlit blocks on top of Hawita special mix (peat, clay, coco peat) in the greenhouse of INOQ GmbH without additional light. Plant nutrition was ensured via a drip irrigation system supplying YaraTera Kristalon Weismarke fertilizer. Half of the plants were inoculated with 1 g/l substrate of the commercial mycorrhiza inoculum INOQ Advantage. RNA isolation was performed using the RNeasy Plant Mini Kit (Qiagen) according to the supplier's instructions followed by routine DNase treatment for red and rigorous treatment using 2 µl DNase and diluted RNA for green fruits using DNA-freeTM Kit (Thermo Scientific). RNA quality was checked by Agilent Bioanalyzer 2100 following supplier´s instructions. Library construction was done by NOVOGENE: After the QC procedures, mRNA was enriched using oligo(dT) beads.The mRNA was then fragmented randomly in fragmentation buffer, followed by cDNA synthesis using random hexamers and reverse transcriptase. After first-strand synthesis, a custom second-strand synthesis buffer (Illumina) was added with dNTPs, RNase H and Escherichia coli polymerase I to generate the second strand by nick-translation. The final cDNA library was ready after a round of purification, terminal repair, A-tailing, ligation of sequencing adapters, size selection and PCR enrichment.