JIA-2019-11

2591 BI Yu-lin et al. Journal of Integrative Agriculture 2019, 18(11): 2589–2597 primers in a final volume of 50 μL. Optimal PCR conditions were 94°C for 5 min (preliminary denaturation), 30 cycles of 94°C for 30 s (denaturation), 50°C for 30 s of primer annealing, 72°C for 30 s (extension), and a final extension at 72°C for 10 min. 2.4. RNA sequencing and identification of lncRNA The RNA-seq was performed in the previous research (Bi et al . 2018). Total RNA was isolated from REV infected or uninfected cells using Trizol Reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. If transcripts were detected in fewer than two experiments, or comprised of a single exon and a length less than 200 bp with a coverage less than 3, or similar to or the same as known porcine small RNAs, or did not pass any analyses of Coding Potential Calculator, Coding-Non-Coding-Index, and Pfam Software, these transcripts would been removed. 2.5. Prediction of target genes Based on the genome location of the lncRNAs and protein- coding genes, the protein-coding genes were identified as target genes located upstream or downstream of lncRNA (within 10 and 100 kb, respectively). The nearest protein- coding genes to lncRNAs with very similar expression patterns were mapped in Gene Ontology (GO) terms of using the GOEAST Software toolkit. The significance level of GO term enrichment was set as an FDR-adjusted p -value<0.05 by the Yekutieli method. 2.6. Kyoto encyclopedia of genes and genomes (KEGG) analysis KEGG pathway information was used in this analysis. Based on the differentially expressed protein-coding genes, the enriched KEGGpathways were identified by a hypergeometric test using the R package ( P <0.01, FDR adjusted). Pathways with fewer than three known chicken genes were discarded. Graphical pathway maps were downloaded from the KEGG FTP server, and differentially expressed protein-coding genes were highlighted according to the coordinate description in the XML files at the KEGG FTP server using Perl GD, XML::Parser and XML::LibXML modules. Table 1 The specific primers for PCR and real-time quantitative PCR (Q-PCR) in this study Gene Sequence Product size (bp) LTR F: 5´-GGCGAGCATCAGACCACT-3´ R: 5´-AGCCTACACCACGAACAA-3´ 288 ENSGALG00000011530 F: 5´-CCTTGTCCAGGGTCCTTACG-3´ R: 5´-GCACAAAAGGGGAGGAGCTA-3´ 209 ENSGALG00000009863 F: 5´-CAAGCAGCGAGTACCCAGAA-3´ R: 5´-GGCAGAGCACAAGACTTCCT-3´ 219 ENSGALG00000005010 F: 5´-ATGTCCAACAGCAGGACTGG-3´ R: 5´-CCGTCAGTCGATCACAAGCA-3´ 119 ENSGALG00000009536 F: 5´-ACAGGAATTCCATGCCGGAG-3´ R: 5´-GGTACCTCACCTGCCCAGT-3´ 148 ENSGALG00000010456 F: 5´-ACCAGCATCACTGTGGACCT-3´ R: 5´-GAAAGGAAGGGAAAAGCAGGGC-3´ 150 ENSGALG00000021171 F: 5´-GGTGACATGAGGTGAGGCAA-3´ R: 5´-TATATTGTGGGGTTCGGGGC-3´ 150 ENSGALG00000025895 F: 5´-GCACAGGTAGAGGCAACTCC-3´ R: 5´-TGAAAAGGCCACCTGTAGAGC-3´ 246 ENSGALG00000011717 F: 5´-GGCTATGGGTGCAGCTTAGAA-3´ R: 5´-CAACACCCCCTGTCTGTGTG-3´ 139 ENSGALG00000010225 F: 5´-AAGAGCCAATGGGGAACAGG-3´ R: 5´-AGACACTCAGTGAACCCCCT-3´ 215 ENSGALG00000014171 F: 5´-TGTTTCAGGCGTGTGAGTGA-3´ R: 5´-GTCCCGCTCAGTGAAATCCA-3´ 161 ENSGALG00000013137 F: 5´-TCAGTGGAAACAGCCGTCAA-3´ R: 5´-TGGGGTGGACTTGAGGTGAA-3´ 208 ENSGALG00000026671 F: 5´-ACCAGTTCTGCACCTTCGTG-3´ R: 5´-CACAGCAACGGTGTGAGATG-3´ 183 NOD1 F: 5´-GCACAGAAGACAGAGTGCCT-3´ R: 5´-AGCAATGCAGGATGGAGGAC-3´ 193 TRAF5 F: 5´-GCTACAGATTTCAGGCGAGG-3´ R: 5´-GGTGTTAAAAGACCCAAAGCCTG-3´ 154 β-Actin F: 5´-TATTGCTGCGCTCGTTGTTG-3´ R: 5´-TGGCCCATACCAACCATCAC-3´ 135