RNA-seq of peripheral whole blood samples comparing obese and non-obese individuals upon inoculation with yellow fever virus vaccine (YF17D)
RNA-seq of peripheral whole blood samples comparing obese and non-obese individuals upon inoculation with yellow fever virus vaccine (YF17D)
Whole blood samples were collected in Tempus blood RNA tubes (Thermo Fisher Scientific, USA). Tempus tubes were stored at -80deg until RNA extraction. RNA extraction from whole blood was performed using the Tempus Spin RNA Isolation kit (Thermo Scientific). RNA was eluted in 90ul of RNase-free water. 1 Î¼g total RNA was used for following library preparation. The poly(A) mRNA isolation was performed using Oligo(dT) beads. The mRNA fragmentation was performed using divalent cations and high temperature. Priming was performed using Random Primers. First strand cDNA and the second-strand cDNA were synthesized. The purified double-stranded cDNA was then treated to repair both ends and add a dA-tailing in one reaction, followed by a T-A ligation to add adaptors to both ends. Size selection of Adaptor-ligated DNA was then performed using DNA Clean Beads. Each sample was then amplified by PCR using P5 and P7 primers and the PCR products were validated.