title: transcriptomic sequencing
design: This project aims to explore the genetic diversity of ticks through de novo transcriptome sequencing. Initially, polyA-enriched mRNA is isolated from tick samples using Oligo(dT) magnetic beads, followed by fragmentation to approximately 300 base pairs (bp) using ion-based fragmentation technology to optimize the junction sequence ratio and sequencing efficiency. Subsequently, double-stranded cDNA is synthesized using random hexamer primers and reverse transcriptase with RNA as the template. After library construction, cDNA fragments are enriched through PCR amplification, and libraries of approximately 450bp are selected. The quality of the libraries is ensured to meet sequencing requirements by conducting a quality check with the Agilent 2100 Bioanalyzer. Then, the total and effective concentrations of the libraries are measured, and libraries with different Index sequences are mixed in proportion according to the required data volume for each sample. The pooled libraries are diluted to 2nM and subjected to alkaline denaturation to form single-stranded libraries in preparation for sequencing. Finally, the libraries are sequenced using the Illumina NovaSeq 6000 platform with Paired-end (PE) sequencing. Data analysis will include initial quality control, alignment, assembly, and analysis to reveal the diversity of gene expression in ticks under different bacterial treatments. The experimental design includes three biological replicates to ensure the reproducibility and reliability of the results.