RNA-seq of Citrus trifoliata : tetraploid roots
Leaves and roots of the 4 citrus rootstock genotypes, control and stressed for the 90mM stress modality, were ground to a fine powder in liquid nitrogen under "RNAse free" condition. RNA extraction was performed using the NucleoMag kit (Macherey-Nagel GmbH &Co.KU, Dren, Germany) with the Kingfisher automated system (KingFisher Flex Purification System (ThermoFisher Scientific, Waltham, MA) according to the manufacturer's protocol. RNA integrity and quality were checked on TapeStation, screentape D5000. RNAseq libraries were prepared to obtain labelled and matched sequences using UDI indexes, following the protocol and recommendations from the Illumina Truseq kit. The integrity and quality of the libraries were checked on TapeStation, screentape D5000 and assayed by qPCR. A first sequencing of the libraries was then performed to validate their quality using the Illumina MiSeq sequencing system at the Agap genotyping platform (Cirad, Montpellier). The equimolar pool of the libraries was then built up using the TruSeq Stranded mRNA Sample Preparation kit from Illumina. Sequencing was performed on a 150 nt pair-end S4 flow cell lane byMGX ( in Montpellier. The clustering and sequencing steps were performed on an Illumina NovaSeq 6000 using the SBS (Sequence By Synthesis) technique with the NovaSeq Reagent Kits using the Illumina TruSeq RNA protocol (Illumina Inc., San Diego, CA, USA).