Stage-wise sequencing of the Meloidogyne incognita trancriptome
To obtain high-throughput transcriptome data of M. incognita, we used Illumina-based NGS sequencing. RNA was extracted from nematodes collected at 5 developmental stages, i.e. egg, J2, J3, J4 and Fe (Female) with three replicates at each stage. Total RNA from the samples was quantified using Nanodrop spectrophotometer (Thermo Scientific), quality-assessed by RNA 6000 Nano assay kit (Agilent) and Bioanalyser2100 (Agilent). NGS sequencing libraries were generated from one microgram of total RNA using Truseq RNA Sample Prep Kit (Illumina) according to the manufacturer's protocol. In brief, the poly-A containing RNA molecules were purified using poly-T oligo attached magnetic beads. After purification, the total poly A+ RNA was fragmented into small pieces using divalent cations under elevated temperature. The cleaved mRNA fragments were reverse transcribed into first strand cDNA using random primers. Short fragments were purified with a QiaQuick PCR extraction kit and resolved with EB buffer for end reparation and addition of poly (A). Following this, the short fragments were connected with sequencing adapters. Each library was separated by adjoining distinct MID tag. The resulting cDNA libraries were then paired-end sequenced (2x101bp) with Illumina HiSeq?? 2000 system.