title: Anadara sativa untreated
design: Total RNA was extracted from the samples in accordance with the manufacturer's instructions using TRIzol reagents (Invitrogen, USA), and RNase-free DNase I (TaKaRa, Japan) was added to remove the DNA contaminants. The degradation and contamination of the extracted RNA was monitored using gel electrophoresis on a 1 % agarose gel. A total of 1.5 g of RNA per sample was used as input material for the RNA sample reparations. Sequencing libraries were generated using the NEBNext Ultra RNA Library Prep Kit for Illumina (NEB, USA) according to the manufacturers recommendations, and index codes were added to attribute the sequences to each sample. Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperatures in NEBNext First Strand Synthesis Reaction Buffer (5X). First-strand cDNA was synthesized using a random hexamer primer and M-MLV Reverse Transcriptase. Second-strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. The remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of the 3 ends of DNA fragments, NEBNext Adaptors with hairpin loop structures were ligated to prepare for hybridization. To select cDNA fragments of preferentially 150~200 bp in length, the library fragments were purified using the AMPure XP system (Beckman Coulter, Beverly, USA). Then, 3 l of USER Enzyme (NEB, USA) was used with size-selected, adaptor-ligated cDNA at 37 C for 15 min followed by 5 min at 95 C before PCR. PCR was performed using Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. Finally, the PCR products were purified (AMPure XP system), and the library quality was assessed on the Agilent Bioanalyzer 2100 system. The clustering of the index-coded samples was performed on a cBot Cluster Generation System using the TruSeq PE Cluster Kit v3-cBot-HS (Illumia, USA) according to the manufacturers instructions. After cluster generation, the library preparations were sequenced on an Illumina Hiseq 4000 platform and paired-end reads were generated.
identifiers.namespace: SUB6578242
identifiers.id: As1