miRNA-seq of I. ricinus
We used the same starting material (total RNA) as in (Kotsyfakis M et al. Tissue- and time-dependent transcription in Ixodes ricinus salivary glands and midguts when blood feeding on the vertebrate host). Briefly, 1,080 nymphs and 420 adult females and males were attached to experimental animals for feeding. Total RNA was extracted from pools of tissues dissected from female adult ticks and nymphal ticks feeding for 3 hour periods up to 24 hours (nymphs) or 36 hours (adults), providing ten samples as follows: four samples for 0Đ12 h and 12Đ24 h for nymphal salivary glands (SG) and midguts (MG) respectively, and six samples for 0Đ12 h, 12Đ24 h, and 24Đ36 h for adult SG and MG respectively. Tick saliva was produced as already described in (Horka H, et al. Tick saliva affects both proliferation and distribution of Borrelia burgdorferi spirochetes in mouse organs and increases transmission of spirochetes to ticks. Int J Med Microbiol. 2009 ). The samples were prepared and sequenced using a 36 bp single-end standard sequencing protocol on a Genome Analyzer II x (Illumina) per the instructions of the manufacturer. The sequencing libraries were prepared according the manufacturerŐs instructions ŇTrueSeq Small RNA Sample Prep GuideÓ. Cluster generation was performed using the Illumina cluster station. The fluorescent images were processed to sequences using the Illumina Genome Analyzer Pipeline Analysis software 1.8