Ampure xp technology

According to the reported protocol, six RNA libraries for input data were generated using NEBNext® Ultra™ RNA Library Prep Kit for Illumina® (NEB, Ipswich, MA, USA). Poly-T oligo-attached to magnetic beads were used to extract mRNA from the prepared RNA library. In the NEBNext First Strand Synthesis Reaction Buffer, fragmentation was performed five times with divalent cations at a high temperature. M-MuLV Reverse Transcriptase and random hexamer primers were used to make first-strand cDNA (RNase H-). Following that, DNA polymerase I and RNase H were used to synthesize second-strand cDNA. The library fragments were purified using the AMPure XP technology to select cDNA fragments ranging from 150 to 200 bp (Beckman Coulter, Beverly, USA). Before PCR, 3 μl of USER Enzyme (NEB, Ipswich, MA, USA) was used with size-selected, adaptor-ligated cDNA at 37°C for 15 minutes and then 5 minutes at 95°C. Phusion High-Fidelity DNA Polymerase, universal PCR primers, and index (X) primer were used in the PCR. According to the manufacturer's instructions, the prepared library quality was assessed using the Agilent Bioanalyzer 2100 system, and PCR products were purified (AMPure XP system). The TruSeq PE Cluster Kit v3-cBot-HS (Illumina) was used to cluster the index-coded samples on a cBot Cluster Generation System.

The TRIzol reagent (Dalian, China) was used to extract the total RNA from 18 hepatopancreas samples at 6, 12, and 24 hpi in accordance with the manufacturer’s instructions. The challenge groups were named AV6_1, AV6_2, AV6_3, AV12_1, AV12_2, AV12_3, AV24_1, AV24_2, AV24_3, whereas the control groups were marked as C6_1, C6_2, C6_3, C12_1, C12_2, C12_3, C24_1, C24_2, C24_3, separately. These libraries were obtained after the whole RNA was first screened and purified using the AMPure XP technology (Beckman Coulter, Beverly, CA, USA). Following these libraries’ initial Qubit 2.0 quantification, they were diluted to 1.5 ng/uL and identified using an Agilent 2100 bioanalyzer. These libraries’ quality was further ensured by using qRT-PCR to precisely measure their effective concentration. The libraries’ construction and sequencing were completed using the Illumina NovaSeq 6000.