GSM9697889: EL24_T1,24h,treatment group1; Ctenopharyngodon idella; RNA-Seq
Total RNA quality assessment: RNA concentration and purity were determined using a NanoDrop 2000 spectrophotometer (Thermo Scientific, Waltham, MA, USA). RNA integrity was evaluated by agarose gel electrophoresis specific for RNA or an Agilent 2100 Bioanalyzer with an RNA 6000 Nano Kit (5067‑1511, Agilent Technologies Inc., Santa Clara, CA, USA). Total RNA with a quantity ≥ 1 μg was used for library preparation. Libraries were constructed using the NEBNext Ultra II RNA Library Prep Kit for Illumina (New England Biolabs Inc., Ipswich, MA, USA), a strand-specific library kit (NEBNext Ultra Directional RNA Library Prep Kit for Illumina). Poly(A)-tailed mRNA was enriched using Oligo(dT) magnetic beads, followed by random fragmentation of mRNA using divalent cations under elevated temperature.Fragmented mRNA was used as a template for first-strand cDNA synthesis with random oligonucleotides. Double-stranded cDNA was purified, followed by end repair, 3′-end adenylation, and ligation of sequencing adapters. cDNA fragments of approximately 400–500 bp were size-selected using AMPure XP beads, then amplified by PCR. PCR products were further purified using AMPure XP beads to obtain the final library.Library quality was assessed using an Agilent 2100 Bioanalyzer with the Agilent High Sensitivity DNA Kit (5067-4626, Agilent Technologies Inc., Santa Clara, CA, USA). Library concentration was quantified using the Quant-iT PicoGreen dsDNA Assay Kit (P7589, Invitrogen, Carlsbad, CA, USA) on a Quantifluor-ST fluorometer (E6090, Promega, Madison, WI, USA). The effective library concentration was determined by qPCR using a StepOnePlus Real-Time PCR System (Thermo Scientific, Waltham, MA, USA).