RNA-Seq of Tunga penetrans
Total RNA was extracted from dissected tissues using the Trizol method (Rio et al., 2010). First-strand complementary DNA (cDNA) synthesis was performed using Superscript II reverse transcriptase (Invitrogen, cat. 18064014) and oligo (dT) primers (Invitrogen, cat. AM1710), followed by second strand synthesis using the Klenow fragment (New England Biolabs, Ipswich, US, cat. M0210S). The double stranded DNA was normalized to 10 ng/L as part of quality control protocols prior to library preparation. Sequencing libraries were constructed using the Nextera library preparation kit (Illumina, San Diego, US, cat. FC-121-1031), which employs a tagmentation step to fragment DNA and ligate adapters simultaneously. PCR amplification was then conducted to enrich libraries and incorporate dual-indexing barcodes. To ensure library integrity, fragment size distribution and quality were assessed using a Bioanalyzer 2100 (Agilent Technologies, Santa Clara, US), confirming a target range of 300700 bp suitable for sequencing. Libraries were sequenced on an Illumina MiSeq platform with 150 bp paired-end chemistry.