RNA-Seq of Dermacentor variabilis: unfed, virgin adult male 4th legs
Poly(A) mRNAs were isolated, separately, from 2  g of total RNA collected from the 1st legs, and 2  g of total RNA collected from the 4th legs for library preparation and barcoding. The mRNAs were fragmented and cDNA synthesized, amplified, digested, and purified utilizing Illumina TruSeq chemistry protocols (Illumina, San Diego, CA, USA). The purified cDNA fragments generated were ligated with adaptor constructs creating the 1st and 4th leg cDNA libraries. In preparation for sequencing, the 1st and 4th leg cDNA libraries were barcoded with a 6 bp barcode (GCCAAT or CTTGTA) to distinguish the sequencing data generated from each library. The barcoded 1st and 4th leg cDNA libraries were hybridized onto one Illumina Hiseq flow cell for cBOT (Illumina, San Diego, CA, USA) cluster generation and sequencing (paired-end, 2   100 bp; total 200 cycles). Two raw data sets of sequencing outputs, one per library, were generated using the Illumina software assembler (Illumina, San Diego, CA, USA)