title: GSM7111185: leaf, treatment with fungal pathogen, rep2 [Cl_PTM2]; Citrus x limon; RNA-Seq
library_construction_protocol: On the seventh day after inoculation, three leaves were taken for each thesis (washed with RNase-free H2O to eliminate the excess of bacteria on the surface). The collected leaves were immediately frozen in situ with liquid nitrogen and stored at -80°C until DNA extraction. DNA extraction was performed with the CTAB protocol (Springer, 2010) after powdering the frozen samples with mortar and pestle using liquid nitrogen. On the seventh day after inoculation, three leaves were taken for each thesis (washed with RNase-free H2O to eliminate the excess of bacteria on the surface). The collected leaves were immediately frozen with liquid nitrogen in situ and stored at -80°C. RNA was extracted using the RNeasy Plant Mini Kit (Qiagen, Venlo, The Netherlands) according to the manufacturer's instructions as detailed in (Russo et al. 2021). 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.