NextSeq 500 sequencing; Total RNA RNAseq from roots of Arabidopsis Col-0 and siz1-2, infected and non-infected with Heterodera schachtii
Total RNA RNAseq from roots of Arabidopsis Col-0 and siz1-2, infected and non-infected with Heterodera schachtii
Complete root systems of 18 plants were excised from leaves, combined into a single tube and snap-frozen in liquid nitrogen. 3 biological replicates per sample were generated. Seeds were vapour sterilised and vernalised at least 3 days before germination. Germination and growth were performed at 21 degrees Celsius with a 16/8 hours light/dark cycle, in 6-well plates with Knop's modified medium. Half of the plants was inoculated with 180 surface-sterilised stage 2 juveniles of H. schachtii, suspended in 0.7% gelrite. Mock infection was done with the same volume of gelrite as required to reach 180 juveniles from the nematode suspension. Plants were  infected 14 days post-germination, roots were harvested at day 21.   Root systems were pulverized with a magnetic bead in a high-speed shaker. RNA was extracted from pulverized tissue using a Maxwell® 16 and the suppliers kit for Plant RNA extraction. Sample preparation was performed using the \"NEBNext Ultra Directional RNA Library Prep Kit for Illumina\" according to the  manufacturer's instructions. Briefly, mRNA was isolated from total RNA using oligo-dT magnetic beads. mRNA was fragmented and cDNA synthesis was performed, subsequently products were ligated to sequencing adapters followed by PCR amplification. The quality and yield after sample preparation was measured with the Fragment Analyzer. The size of the resulting products was consistent with the expected size distribution (a broad peak between 300-500 bp).