alias: GSM2810008
title: GSM2810008: D42_Con RNA-seq; Paralichthys olivaceus; RNA-Seq
library_construction_protocol: Total amounts of 3 μg RNA per sample were used for sequencing libraries via the NEBNext UltraTM RNA Library Prep Kit for Illumina (NEB, USA) following the manufacturer's recommendations. Briefly, mRNA was purified with poly-T oligo-attached magnetic beads and fragmented using divalent cations under elevated temperature in the NEBNext First Strand Synthesis Reaction Buffer (5X). Then, the first and second strand cDNA were separately synthesized using random hexamer primer and M-MuLV reverse transcriptase (RNase H-), DNA polymerase I, and RNase H. After adenylation of the 3' ends of DNA fragments, a NEBNext Adaptor with hairpin loop structure was ligated to prepare for hybridization. To select cDNA fragments of preferentially 150-200 bp in length, library fragments were purified with the AMPure XP system (Beckman Coulter, Beverly, USA). Then, 3 μl USER Enzyme (NEB, USA) was incubated with size-selected, adaptor-ligated cDNA at 37°C for 15 min followed by 5 min at 95°C prior to PCR. The subsequent PCR was performed with Phusion High-Fidelity DNA polymerase, universal PCR primers, and Index (X) Primer. Finally, PCR products were purified (with the AMPure XP system) and the library was assessed on the Agilent Bioanalyzer 2100 system and sequenced on the Illumina Hiseq 4000 platform, generating 150 bp paired-end reads. RNA libraries were prepared for sequencing using standard Illumina protocols
identifiers.namespace: GEO
identifiers.id: GSM2810008