Feed-microbiome-host interactions in Atlantic salmon over life stages
RNA extractions from pyloric caeca and hindgut samples obtained from fish for the high dose -mannan trial were conducted using the RNAdvance Tissue kit (Beckman Coulter, Inc.) following the manufacturer's protocol (PN B66716AD). RNA concentration and purity were determined using a Qubit 3.0 fluorometer following the manufacturers protocol. RNA integrity was checked by using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Samples with a RIN (RNA integrity number) equal to or above two were used. Before analysis, all samples were randomized. For both the trials, the library preparations were carried out by Novogene (Beijing, China) using a TruSeq Stranded mRNA kit (Illumina, San Diego, CA, USA), as per the manufacturers protocol. Briefly, polyA-containing mRNA molecules were isolated, using magnetic oligo dT beads, from the total RNA according to the polyA selection method and then fragmented with the fragmentation buffer. Cleaved RNA fragments were primed with random hexamers into first-strand cDNA using reverse transcriptase and random primers. After a double-stranded cDNA was synthesized, followed by end-repair, phosphorylation and A base addition, and adapter ligation according to Illuminas library construction protocol. Libraries were sequenced on the Illumina NovaSeq 6000 platform at Novogene, (Beijing, China), using 300 bp paired-end sequencing.