Instrument: Illumina NovaSeq 6000
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: cDNA
Layout: PAIRED
Construction protocol: At 1, 2, 3, 4 and 10 days after infection all chickens in one of the infected groups and one or two of the uninfected control chickens were killed by cervical dislocation and the two caeca were collected. Approximately 1 cm of the proximal part of the caeca containing the caecal tonsils was cut off. The remaining caeca were cut open longitudinally and caecal contents were thoroughly rinsed off with ice cold PBS. Ceacal tissues were cut into 0.5 cm wide strips and the tissue from the two caeca from each bird where pooled in 5 ml RNAlater (RNAlater® stabilization solution, Invitrogen, ThermoFisher Scientific). Samples in RNAlater were subsequently stored at 4°C for 24 h and thereafter stored at -20°C until RNA isolation. Caecal tissues stored in RNAlater were thawed, removed from the solution and briefly air-dried. Tissues were then added to test tubes containing 10 ml of TRIzol and a 1:1 mixture of 2 mm ø and 0.5 mm ø XX beads (XX). Tubes were placed in a XX and shaken three times for 60 s at 6.2 m/s. After this treatment all mucosal tissue but not all connective tissue was dissolved. Samples were then stored at – 70°C until RNA isolation. For RNA isolation 1 ml from each of the caecal samples homogenised in TRIzol was used and RNA was extracted using chloroform according to the TRIzol manufacturer's RNA isolation protocol. The Isolated RNA was subsequently treated with DNase (TURBO™ DNase, 2U/ul, ThermoFisher Scientific) according to the manufacturers protocol (Pub. No. MAN0001271) and further purified using reagents and the “RNA cleanup” protocol of the RNeasy Mini kit (Qiagen) protocol. RNA concentration and quality was then assessed using the Agilent RNA 6000 Nano kit on a 2100 Bioanalyzer Instrument (Agilent) and the RNA stored at -70°C until further analysis. Sequencing libraries were prepared from 500ng total RNA using the TruSeq stranded mRNA library preparation kit (Cat# 20020594/5, Illumina Inc.) including polyA selection.The library preparation was performed according to the manufacturers' protocol (#1000000040498). NovaSeq 6000 S4 flowcell, paired-end 150bp read length, v1 sequencing chemistry.