NGS-RNA-Seq of Phthonandria atrilineata: adult mussle
Total RNA was extracted from the muscle tissue of adult Phthonandria atrilineata specimens. The quality and integrity of the extracted RNA were assessed using an Agilent 2100 Bioanalyzer, ensuring an RNA Integrity Number (RIN) > 8.0 for library preparation. The RNA-Seq library was prepared using the NEBNext Ultra II RNA Library Prep Kit for Illumina (New England Biolabs) according to the manufacturer's protocol. Briefly, mRNA was isolated from 1 g of total RNA using oligo-dT magnetic beads, followed by fragmentation using divalent cations under elevated temperature. First-strand cDNA synthesis was performed using random primers and SuperScript III reverse transcriptase. Second-strand cDNA synthesis was then carried out using DNA Polymerase I and RNase H. The resulting double-stranded cDNA was subjected to end-repair, A-tailing, and adapter ligation with Illumina-compatible adapters. The adapter-ligated DNA was then purified and size-selected using AMPure XP beads to obtain an average insert size of 300-400 bp.Library amplification was performed using random PCR with Illumina-specific primers for 12-15 cycles to enrich for properly ligated fragments and to add index sequences for multiplexing. The final library was purified using AMPure XP beads. The quality of the prepared library was assessed using an Agilent 2100 Bioanalyzer, and the quantity was determined by qPCR using the KAPA Library Quantification Kit (Kapa Biosystems). The final library was then sequenced on an Illumina NovaSeq 6000 platform using paired-end 150 bp sequencing chemistry, following Illumina's standard protocols.
SUB14765118
PHAT_RNA_2024