2.2. RNA Extraction, Library Preparation and Sequencing

Whole blood samples were collected from the 51 volunteers in the high dose group, vaccinated with either 10⁷ or 5 × 10⁷ PFU of rVSVΔG-ZEBOV-GP, and from the 13 placebo. Blood samples were taken at day 0, day 1, day 3, day 7, day 14, day 28, day 35, and day 168 post immunization and stored at −80 °C in PAXgene Blood RNA kit (PreAnalytiX, QIAGEN, Hombrechtikon, Switzerland). Total RNA extraction was performed using PAXgene blood RNA Kit (PreAnalytiX, QIAGEN), according to manufacturer’s protocol (PAXgene Blood RNA kit Handbook, version 2, 2015). After extraction, RNA was quantified using the Qubit^® 2.0 Fluorometer with the RNA HS Assay Kit (Thermo Fisher Scientific, Waltham, MA, US).

Moreover, 50 ng of total RNA was reverse transcribed to produce cDNA, using SuperScript™ VILO™ cDNA Synthesis Kit (Thermo Fisher Scientific). cDNA was used to prepare the libraries with the Ion AmpliSeq™ Transcriptome Human Gene Expression Kit (Thermo Fisher Scientific), according to manufacturer’s recommendation, this kit allows the amplification of 20,812 human genes following 10 cycles of PCR amplification. Libraries were barcoded with Ion Xpress™ Barcode Adapters in order to sequence eight samples on each chip, the barcoded libraries were purified using Agencourt AMPure XP Magnetic Beads (Beckman Coulter, Brea, CA, US). The quantity of each library was evaluated using Ion Library TaqMan Quantitation Kit (Thermo Fisher Scientific) on a ViiA 7 Real-Time PCR System (Thermo Fisher Scientific).

Libraries were diluted to a concentration of 40 pM and pooled in groups of eight for sequencing on chips (Ion PI Chip Kit v3, Thermo Fisher Scientific), which were loaded using Ion Chef System (Thermo Fisher Scientific) and the IonPI Hi-Q Chef Kit (Thermo Fisher Scientific) according to manufacturer’s protocol. Sequencing reactions were performed on an Ion Proton Sequencer (Thermo Fisher Scientific) using Ion PI Hi-Q Sequencing 200 Kit (Thermo Fisher Scientific).