title: 454 Sequencing of Dermacentor variabilis EST Library
design: American dog ticks, D. variabilis, were reared as previously described (Sonenshine, 1993) and originated from specimens collected near Richmond, Virginia, USA.  Adult ticks were confined within plastic capsules attached to New Zealand white rabbits, Oryctolagus cuniculus, and allowed to feed as required for experiments described below.  Larvae and nymphs were fed on Norway rats, Rattus norvegicus.  Rearing conditions were 26 ± 1°C, 92 ± 6% relative humidity and 14:10 (L:D).  Approximately 50 synganglia for each developmental condition, including lateral secretory organs and pedal nerves were dissected from female D. variabilis that were either unfed, part-fed virgin (attached to the host for 4-5 d), part-fed virgin forcibly detached from the rabbit host and held in culture for 4-5 d, part-fed mated (allowed to mate for =1 d) or replete (1 d post drop off from the host).  Tissues from each feeding stage were separately homogenized in TRI Reagent (Sigma-Aldrich, St. Louis, MO) according to the manufacturer’s recommendations.  RNA pellets were rehydrated in 100 µM aurintricarboxylic acid to prevent degradation (Hallick et al., 1977).  Approximately 3 µg of total RNA from each group were pooled, and mRNA was isolated using an Oligotex mRNA isolation kit (Qiagen, Valencia, CA) according to the manufacturer’s recommendations.  Purified mRNA was ethanol precipitated and rehydrated in 2 µl and combined with 10 picomoles of modified 3’ reverse transcription primer (5’-ATTCTAGAGACCGAGGCGGCCGACATGT(4)GT(9)CT(10)VN-3’) (Beldade et al. 2006) and 10 picomoles SMART IV oligo (5’-AAGCAGTGGTATCAACGCAGAGTGGCCATTACGGCCGGG-3’) (Zhu et al. 2001).  The resulting 4 µl were incubated at 72°C for 2 min and then combined with the following reagents on ice: 1 µl RNase Out (40 U/µl), 2 µl 5X first strand buffer, 1 µl 20 mM DTT, 1 µl dNTP mix (10 mM each) and 1 µl Superscript II reverse transcriptase (Invitrogen, Carlsbad, CA).  The reaction was incubated at 42°C for 90 min then diluted to 30 µl with TE buffer (10 mM Tris HCL pH 7.5, 1 mM EDTA) and stored at -20°C until further use.  To synthesize second strand cDNA, 5 µl of first-strand cDNA was mixed with 10 picomoles of modified 3’ PCR primer (5’-ATTCTAGAGGCCGAGGCGGCCGACATGT(4)GTCT(4)GTTCTGT(3)CT(4)VN-3’) (Beldade et al. 2006), 10 picomoles of 5’ PCR primer (5’-AAGCAGTGGTATCAACGCAGAGT-3’) (Zhu et al. 2001), 5 µl 10X reaction buffer, 1 µl dNTP mix, 2 µl MgSO4, 0.4 µl Platinum HiFi Taq Polymerase and 34.6 µl H2O (Invitrogen).  Thermal cycling conditions were 94°C for 2 min followed by 20 cycles of 94°C for 20 sec, 65°C for 20 sec and 68°C for 6 min.  The first PCR reaction was conducted, and 5 µl aliquots from cycles 18, 22 and 25 were analyzed on a 1% agarose gel to optimize the number of cycles.  An additional 5 reactions were conducted to produce sufficient quantities of cDNA for 454 library preparation.  The contents were combined, and the cDNA was purified by using a PCR purification kit (Qiagen) according to the manufacturer’s recommendations.  The cDNA library was prepared with appropriate kits (Roche, Indianapolis, IN; Qiagen) for pyrosequencing on the GS-FLX sequencer (Roche) according to the manufacturer’s recommendations which were described previously (Margulies et al. 2005).  The only deviation from the protocol was prior to titration sequencing; following emulsification PCR, DNA-positive beads were enriched in order to increase the number of reads collected during titration.