title: GSM2354326: BDE-47 treatment group; Mactra quadrangularis; RNA-Seq
library_construction_protocol: TRIzol reagent (Invitrogen, Carlsbad, CA, USA) was used to extract the total RNA of control and treatment samples following the manufacturer's protocol. After RNase-free DNase treatment (Takara Biotechnology, China), the purity of total RNA was determined using an ultraviolet spectrophotometry, Thermo Scientific NanoDrop 8000. RNA concentration was measured using Qubit®RNA Assay Kit in Qubit®2.0Flurometer (Life Technologies, CA, USA). RNA integrity was checked using the RNA Nano 6000 Assay Kit of the Agilent 2100 Bioanalyzer (Agilent Technologies,CA, USA). cDNA library construction was performed at Beijing Genomics Institute (BGI, Shenzhen, China) using the NEBNext® Ultra™ RNA Library Prep Kit for Illumina® (NEB, USA). Briefly, mRNA was separated from the total RNA using poly-T oligo-attached magnetic beads and then interrupted into small pieces. The short mRNA was reverse transcribed into first-strand cDNA using reverse transcriptase and random hexamer primers. Followed by adding dNTPs, Rnase H, and DNA polymerase I, second strand cDNA was synthesized with first-strand cDNA as the template. Then, the double stranded cDNA was purified with QiaQuick PCR extraction kit (Qiagen, Hilden, Germany), repaired the cohesive, and ligated with sequencing adaptors. After screening the target fragment with agarose gel electrophoresis, PCR amplification was performed using the recovered fragments as the template. The quality and output of cDNA library were tested by Agilent 2100 Bioanalyzer and ABI StepOnePlus Real-time PCR System. Afterwards, library sequencing was performed on an Illumina Hiseq 2000 platform.