Tabanus bromius adult female sialotranscriptome
Salivary glands (SG) were stored in RNAlater at 4°C for 48 h before being transferred to –70° C until RNA extraction. SG RNA was extracted and isolated using the Micro FastTrack mRNA isolation kit (Invitrogen, Grand Island, NY) per manufacturer’s instructions. The integrity of the total RNA was checked on a Bioanalyser (Agilent Technologies, Santa Clara, CA). mRNA library construction and sequencing were done by the NIH Intramural Sequencing Center. The SG library was constructed using the TruSeq RNA sample prep kit, v. 2 (Illumina Inc., San Diego, CA). The resulting cDNA was fragmented using a Covaris E210 (Covaris, Woburn, MA). Library amplification was performed using eight cycles to minimize the risk of over-amplification. Sequencing was performed on a HiSeq 2000 (Illumina) with v. 3 flow cells and sequencing reagents. One lane of the HiSeq machine was used for this and four other libraries, distinguished by bar coding.  Libraries  from the tick Hyalloma annatolicum excavatum, the triatomine Panstrongylus megistus and the bat Diphila ecaudata were co-sequenced with T. bromius and  we found that some cross-contamination of sequences occurred between the libraries.