title: guts of Trioza camphorae. (CRX771164; CRA012312)
library_construction_protocol: Once the integrity and quantity of the RNA were verified, the samples were dispatched to Novogene (Beijing, China) for library preparation and subsequent Illumina sequencing. Briefly, mRNA was isolated from the total RNA using poly-T oligo-attached magnetic beads. The RNA fragments were then fragmented using divalent cations at an elevated temperature in a 5X First Strand Synthesis Reaction Buffer. The initial strand of cDNA was synthesized using a random hexamer primer and M-MuLV Reverse Transcriptase. The resulting RNA was then degraded with RNaseH. Subsequently, the second strand of cDNA was synthesized using DNA Polymerase and dNTP. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adding adenine residues to the 3' ends of the DNA fragments, adaptors with hairpin loop structures were ligated to enable hybridization. To ensure a preference for cDNA fragments ranging from 370 to 420 base pairs in length, the library fragments were purified using the AMPure XP system from Beckman Coulter (Beverly, USA). Then, PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index Primer.