RNA-seq of hard clams under 45 psu seawater for 1 day
mRNA was enriched from total RNA using poly-T oligo-attached magnetic beads. Fragmentation of mRNA was carried out using divalent cations under elevated temperature in first strand synthesis reaction buffer. The random hexamer primer and M-MuLV Reverse Transcriptase were used to synthesize the first strand cDNA. The second strand cDNA was synthesized using DNA Polymerase I and RNase H. After adenylation of 3' ends of DNA fragments, the adaptor with hairpin loop structure were ligated to prepare for hybridization. The library fragments were purified using AMPure XP system (Beckman Coulter, USA), and the cDNA fragments with length of 370420 bp were selected. The PCR was performed using Phusion High-Fidelity DNA Polymerase, universal PCR primers and index primer. The PCR products were purified using AMPure XP system, and library quality was evaluated using Agilent Bioanalyzer 2100 system. The clustering of the index-coded samples was performed on a cBot Cluster Generation System using TruSeq PE Cluster Kit v3-cBot-HS. After cluster generation, the cDNA library was sequenced on an Illumina Novaseq platform, and 150 bp paired-end reads were generated.