GENOMIC
Add mRNA Capture Beads to the prepared total RNA, mix thoroughly, and incubate at 65C to denature the RNA. After denaturation, incubate at room temperature for 5 min to allow mRNA to bind to the beads. Place the tube on a magnetic rack until the beads collect against the magnet, then discard the supernatant. Remove the tube from the rack, resuspend the beads in Wash Buffer, return to the rack, and discard the supernatant once the beads have collected. Remove from the rack, add Tris Buffer, mix, and elute the mRNA at 80C. Add Beads Binding Buffer to the tube, mix thoroughly, and incubate at room temperature for 5 min to allow mRNA to rebind to the beads. Place the tube on the magnetic rack until the beads collect, then discard the supernatant. Remove from the rack, wash with Wash Buffer, return to the rack for 2 min, and then discard the supernatant. Remove from the rack, add Frag/Prime Buffer, and mix. Transfer the resulting mRNA to a PCR machine, determining appropriate fragmentation conditions based on sample quality. Add reagents for first-strand reverse transcription to the reaction tube, mix, centrifuge, and run the PCR program to synthesize first-strand cDNA. Sequentially add reagents for second-strand synthesis and end repair to the first-strand cDNA, mix, centrifuge, and run the program. After the reaction, immediately perform adapter ligation. Add adapters and ligase sequentially, and incubate in a metal bath for adapter ligation. Add an appropriate volume of (0.8X) mixed magnetic beads to the reaction, mix thoroughly, incubate at room temperature, then place on the magnetic rack; after the beads collect, discard the supernatant. Remove from the rack, add Wash Buffer, mix, return to the rack, and discard the supernatant after the beads collect. Finally, add Elution Buffer, mix gently, and incubate at room temperature for 5 min. Place the tube on the magnetic rack until the beads collect, then collect the supernatant for subsequent analysis. Perform sequencing of the constructed library using the Illumina NovaSeq 6000.