The trachea tissue of the chicken injected with PBS (3 days post-challenge), replication 3
The RNA was quantified and assessed for purity by a 2100 Bioanalyzer (Agilent Technologies, Waldbronn, Germany). A sequencing library was prepared with 1g of total RNA for each sample using the TruSeq Stranded Total RNA with LT Sample Prep Kit (Illumina, San Diego, CA, USA). Briefly, to deplete bacterial and eukaryotic ribosomal RNA (rRNA), the total RNA was treated with the Ribo-Zero rRNA Removal Kit (Epicentre, Madison, WI, USA). The cDNA synthesis was performed using reverse transcriptase and random hexamers and the second strand was synthesized as recommended by manufacturer. These cDNA fragments underwent an end repair process, the addition of a single A base, and ligation of the indexing adapters. The products were purified and enriched with PCR to create the final cDNA library. The constructed libraries were quantified and qualified using a qPCR quantification protocol guide (TruSeq Stranded Total RNA Sample Prep Guide, Part # 15031048 Rev. E). The resulting cDNA libraries were sequenced using the Illumina NovaSeq platform, generating approximately 769 million paired-end reads of 101 nucleotides in length.