Fig. 1

Experimental design, sampling and data acquisition during in vitro culturing of Haemonchus contortus larvae. Schematic representation of the in vitro culture system, developmental transition from the free-living, infective stage (exsheathed L3, xL3) to the early parasitic, blood-feeding stage of H. contortus, sampling strategy, and transcriptomic/proteomic analyses. Infective third-stage larvae (xL3s) are obtained by exsheathment (0.15% sodium hypochlorite, 38 °C) and cultured for 72 h at 38 °C under 10% CO₂ in control medium or medium supplemented with 100 µM haemin chloride, 10% heat-inactivated sheep serum, or 10% heat-inactivated defibrinated sheep blood. Total RNA is extracted from cultured larvae for cDNA synthesis and subsequent Illumina RNA sequencing. Raw sequencing reads are processed by adapter trimming, quality filtering (FastQC), alignment to the H. contortus reference genome, and gene-level quantification. Differential gene transcription analysis is performed using DESeq2. Cultured larvae are also processed for protein isolation, digestion and LC-MA/MS analysis. Mass spectrometry data is processed using Proteome Discoverer and MaxQuant. Technical validations are performed via daily visual inspection of cultured larvae to confirm viability and sterility; box-plot analysis of log₂(FPKM + 1) values across libraries to assess data distribution and normalization; gene count thresholds (FPKM > 1) to evaluate transcriptome coverage; hierarchical clustering and t-SNE to confirm biological reproducibility and treatment-specific expression patterns; and differential gene expression analysis using volcano plots (|log₂FC| > 1, adjusted P < 0.05). These metrics confirm high data quality, low technical bias, and clear transcriptional responses suitable for integrative transcriptomic and proteomic analyses.