RNA-Seq of Citrus x limon
Mock and CLas-infected leaves were used for total RNA extraction as per the manufacturers instructions (Invitrogen, CLD, CA). The RNA content was measured after the elimination of genomic DNA using DNase I (Takara, Japan). The RNA integrity number (RIN6.5) was evaluated using the Bioanalyzer 2100 (Agilent) and subsequently quantified using the ND-2000 (NanoDrop Technologies). Samples with high RNA quality (OD260/280=1.8~2.2, OD260/2302.0, RIN6.5, 28S:18S1.0, >10g) were used for sequencing library construction by using the TruSeq Small RNA Library Prep Kit. RNA-seq libraries were generated following the TruSeqTM RNA sample preparation kit (Illumina, San Diego, CA). 5 g of RNA was used to create RNA-seq transcriptomic libraries. Messenger RNA (mRNA) was isolated using polyA selection using oligo(dT) beads and subsequently fragmented using a fragmentation buffer. The process of adding and joining the Illumina-indexed adaptors was carried out following the protocol provided by Illumina. The libraries were later chosen based on the size of cDNA target fragments, specifically, those measuring 200-300 bp, using a 2% low-range ultra-agarose. These selected libraries were then subjected to PCR amplification using Phusion DNA polymerase (NEB) for 15 PCR cycles. The TBS380 quantified the samples, and then the Illumina HiSeq4000 sequenced the paired-end libraries.