Deep sequencing of hepatic RNA-seq libraries from blunt snout bream fed high-fat diets
6 µg total RNA from each of pooled samples was used to synthesize double-strand cDNAs using Oligo(dT) primers. After digestion with the restriction enzyme NlaIII, which can recognize and cut off CATG sites, the bead-bound cDNA fragments with intact Oligo(dT)-linked 3’ ends were purified by magnetic bead precipitation, ligated with Illumina adaptor 1 at the produced sticky 5’ ends. The junction of Illumina adaptor 1 and CATG site is designed as the recognition site of MmeI Endonuclease, which cuts the cDNA at 17 bp downstream of the CATG site, producing tags linked with adapter 1. Such tags were subsequently purified by removing 3’ fragments via magnetic bead precipitation and ligated with Illumina adaptor 2 at the sticky 3′ ends of tags to generate a tag library with different adaptors at both ends of tags. After 15 cycles of linear PCR amplification with the adaptor-specific primers, 95 bp fragments are purified by 6% TBE PAGE Gel.