GSM8084794: control 2n_replicate 2; Carassius gibelio; RNA-Seq
Total RNA was isolated from each individually preserved spleen using PureLink® RNA Mini Kit (Ambion) along with TRIzol™ Reagent (Invitrogen) and PureLink® DNase (Ambion). The quantity of extracted RNA was assessed using a NanoDrop 8000 spectrophotometer (Thermo Fisher Scientific). The quality and integrity of RNA were analyzed using RNA 6000 Nano Kit on a 2100 Bioanalyzer instrument (Agilent Technologies). RNA samples with an acceptable RNA integrity number (RIN>7) were used for DNA library preparation. 500 ng of total RNA was used for mRNA enrichment using the Poly(A) mRNA Magnetic Isolation Module (New England Biolabs). Subsequently, NEBNextUltra II DirectionalRNA Library Prep Kit for Illumina, and Dual Index Primers Set 2 of NEBNextMultiplex Oligos for Illumina (New England Biolabs) were used for library preparation. The numbers of PCR cyclers used for enrichment and the beads ratio used for fragment size selection were modified according to the recommendations in the manufacturers´ protocol. The quantification of DNA libraries was performed on a Qubit 4 fluorometer (Invitrogen by Thermo Fisher Scientific) using Qubit dsDNA HS Assay Kit, and quality and size controls were performed on a 2100 Bioanalyzer with DNA 1000Kit (Agilent Technologies). Finally, amplicons (average size of 355 bp) were pooled in equimolar amounts.