title: GSM5736609: RNA sequencing of whole body Trachonitus carolinus larvae: Days Post Hatch D3, Tank P8, Salinity S30, replicate a; Trachinotus carolinus; RNA-Seq
library_construction_protocol: Total RNA was extracted a set of homogenized larval samples using the Qiagen RNeasy Mini Kit and kept at -20°C. All extracts were checked for concentration (ng/µL) using a Qubit Fluorometer 3 (ThermoFisher Scientific, Waltham, MA, USA) and quality (A260/A280) using a ThermoFisher NanoDrop Spectrophotometer 2000. Purified RNA samples were sent to GeneWiz (South Plainfield, NJ, USA) for high-throughput sequencing. Samples were quantified using Qubit 2.0 Fluorometer (Life Technologies, Carlsbad, CA, USA) and RNA integrity was checked using Agilent TapeStation 4200 (Agilent Technologies, Palo Alto, CA, USA). RNA sequencing libraries were prepared using the NEBNext Ultra II RNA Library Prep Kit for Illumina following manufacturer's instructions (NEB, Ipswich, MA, USA). Briefly, mRNAs were first enriched with Oligo(dT) beads. Enriched mRNAs were fragmented for 15 minutes at 94 °C. First strand and second strand cDNAs were subsequently synthesized. cDNA fragments were end repaired and adenylated at 3'ends, and universal adapters were ligated to cDNA fragments, followed by index addition and library enrichment by limited-cycle PCR. The sequencing libraries were validated on the Agilent TapeStation (Agilent Technologies, Palo Alto, CA, USA), and quantified by using Qubit 2.0 Fluorometer (Invitrogen, Carlsbad, CA) as well as by quantitative PCR (KAPA Biosystems, Wilmington, MA, USA). The sequencing libraries were clustered on one flowcell lane. After clustering, the flowcell was loaded on the Illumina HiSeq instrument (4000 or equivalent) according to manufacturer's instructions. The samples were sequenced using a 2x150bp Paired End (PE) configuration. Image analysis and base calling were conducted by the HiSeq Control Software (HCS). Raw sequence data (.bcl files) generated from Illumina HiSeq was converted into fastq files and de-multiplexed using Illumina's bcl2fastq 2.17 software. One mismatch was allowed for index sequence identification.