RNA-Seq of Sus scrofa: weaned piglets spleen during African Swine Fever Virus infection
Total TRIzol (Invitrogen) was used to extract total RNA from the spleen tissue of weaned piglets according to the manufacturers protocol. A Nanodrop 2000 was used to detect the concentration and purity of the extracted RNA, and an Agilent 2100 was used to determine the RIN value. The total amount of sample RNA was1 g, the concentration was 35 ng/L, OD260/2801.8, and OD260/2301.0 to construct a sequencing library. The 3' end of eukaryotic mRNA had a poly A tail structure. Using magnetic beads with Oligo(dT) and poly-A to perform AT base pairing, mRNA was isolated from the total RNA for transcriptome analysis. Fragmentation buffer was added, appropriate conditions were selected, and the mRNA was randomly fragmented to approximately 300 bp. Under the action of reverse transcriptase, random primers were used to reverse synthesise single-stranded cDNA using mRNA as a template, followed by double-stranded synthesis. The double-stranded cDNA structure had sticky ends. Thus, an End Repair Mix was added to make it blunt-ended, and then an A base was added to the 3' end to connect the Y-shaped adapter. After ligation with the adapter, the products were purified and fragments were sorted, which were further used for PCR amplification and purification to obtain the final library. QuantiFluor dsDNA System quantification was used, with a mix according to the data ratio. Bridge PCR amplification was performed on cBot to generate clusters and was sequenced in an Illumina NovaSeq 6000.