GSM1685000: Infected sample, replicate 2b; Ixodes scapularis; RNA-Seq
Purified RNAs were used for library preparation using the TruSeq-based NEB-Next Ultra Directional RNA Library Prep kit (New England Biolabs, Ipswick, MA, USA). Briefly, 1 µg total RNA was used as starting material for library preparation. Messenger RNA was captured using oligo-dT magnetic beads (Poly(A) RNA Magnetic Isolation Module; New England Biolabs) and purified polyA+ RNA was chemically fragmented and reverse-transcribed. A controlled fragmentation period was applied to obtain an insert size of approximately 320 nucleotides, best suited to generate non-overlapping reads after pair-end sequencing. The second strand was generated and dsDNA was purified using AMPure SPRI-based magnetic beads (Beckman Coulter, IZASA, Spain). Next, samples were end-repaired followed by adaptor ligation and removal of excess oligonucleotides by double AMPure selection, made according to NEB-Next recommendations as a function of library size. Adaptor oligonucleotides contained the signals for subsequent amplification and sequencing as well as sample-specific identifiers, which allowed multiplexing in the sequencing run. An enrichment procedure based on PCR was then performed to ensure that all molecules in the library conserved the adapters at both ends. The number of PCR cycles was adjusted to 13 for all four samples. The final amplified library was checked again on a BioAnalyzer 2100 (Agilent, Santa Clara, CA, USA), pooled, quantified by fluorimetric methods (PicoGreen®) and titrated by real-time PCR using a well-controlled library as standard. For RNAseq, libraries were denatured and seeded on the surface of a Pair-End Flowcell where clusters were generated and sequenced using a HiSeq2000 equipment (Illumina, San Diego, CA, USA), under a 2x100 recipe.
GEO Accession
GSM1685000