midgut transcriptome shotgun sequencing from unfed Ixodes ricinus females
Explanted Ixodes ricinus midguts were homogenized in a dounce homogenizer. Total RNA was extracted with TRIzol Reagent (Life technologies, Carlsbad, CA, USA) and DNA was digested with DNAfree kit (Life technologies, Carlsbad, CA, USA) according to the manufacturer’s protocol. RNA was purified with RNAcleanXP beads (BeckmanCoulter, Brea, CA, USA) with a sample to beads ratio of 1:1.8. mRNA was extracted with Dynabeads RNA DIRECT Micro Kit (Life technologies, Carlsbad, CA, USA). I. ricinus midgut whole transcriptome library was prepared with the Ion Total RNA-Seq Kit v2 (life technologies, Carlsbad, CA, USA) according to the manufacturer’s protocol. Sequencing was accomplished on an Ion PGM sequencer with the Ion PGM Template OT2 200 Kit for template preparation and the Ion PGM Sequencing 200 Kit v2 for sequencing on Ion 314TM Chips v2 and Ion 318TM Chips (life technologies, Carlsbad, CA, USA) according to the manufacturer’s protocol.
Luxembourg Institute of Health
midgut_IonTorrent