GSM8711860: male 2n replicate 6; Carassius gibelio; RNA-Seq
Total RNA was isolated using PureLink® RNA Mini Kit (Ambion) with Trizol reagent (Thermo Fisher Scientific, Waltham, MA, USA) and on-column PureLink DNase treatment was used according to the manufacturer´s protocol. Reagent and buffer volumes were adjusted according to the weight of tissue entering the isolation process (30 mg on average). The final elution was performed using 100 µl of RNAse-free water in the first step and the primal eluate in the second step. The yield and concentration of RNA isolates were checked using a QubitTM 4 fluorometer (Invitrogen by Thermo Fisher Scientific, Waltham, MA, USA) and Qubit RNA HS Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA). The quality and integrity of RNA were analysed using RNA 6000 Nano Kit on a 2100 Bioanalyser instrument (Agilent Technologies, Santa Clara, CA, USA). All RNA isolates were normalized by dilution at a uniform concentration of 20 ng/µl with RNase-free water. They served as templates for DNA library preparation in twice the reaction volume recommended by the manufacturer. RNA integrity (RIN > 7) was checked for each sample on the Fragment Analyzer using RNA Kit 15 nt (Agilent Technologies). First, 250-500 ng of total RNA was used for mRNA enrichment using the NEBNext Poly(A) mRNA Magnetic Isolation Module (New England Biolabs). Subsequently, full-length RNA-seq libraries were prepared using NEBNext Ultra II Directional RNA Library Prep Kit (New England Biolabs) with xGen™ CS adapters (IDT) containing UMIs. Quality control for library quantity and size distribution was done using QuantiFluor dsDNA System (Promega) and High Sensitivity NGS Fragment Analysis Kit (Agilent Technologies). Final library pool was sequenced on NovaSeq 6000 (Illumina) in paired-end 150bp mode, resulting in an average of 30 million reads per sample.