title: GSM4996600: Leaf Plenodomus tracheiphylus treated rep2; Citrus jambhiri; RNA-Seq
library_construction_protocol: Both inoculated and control samples were collected 15 days after inoculation. Leaves were immediately frozen with liquid nitrogen and stored at -80°C until both DNA and RNA extractions were performed. DNA extraction was performed according to Springer 2010. The RNA was extracted using the RNeasy® Plant Mini Kit (Qiagen, Venlo, Netherlands) according to the manufacturer's instructions. All samples passed through three steps before library construction: Nanodrop for preliminary quantitation, agarose gel electrophoresis to tests RNA degradation and potential contamination, Agilent 2100 to checks RNA integrity and quantitation. After the QC procedures, mRNA from is enriched from total RNA using oligo(dT) beads. The mRNA is 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) is added, with dNTPs, RNase H and Escherichia coli polymerase I to generate the second strand by nick-translation and AMPure XP beads is used to purify the cDNA. The final cDNA library is ready after a round of purification, terminal repair, Atailing, ligation of sequencing adapters, size selection and PCR enrichment. Libraries are fed into Illumina machines according standard Illumina protocol.