Transcriptome analysis Rhinolophus ferrumequinum (Rfe) cells upon poly-I:C transfection
Transcriptome analysis Rhinolophus ferrumequinum (Rfe) cells upon poly-I:C transfection
Total RNA was extracted from cell lysates using the NucleoSpin RNA II kit (Macherey-Nagel) and eluted in water For polyI:C stimulation, RFe cells were plated in 12-well plates. The next day, they were transfected with 100 ng polyI:C (InvivoGen) or PBS, respectively, using INTERFERin (Polyplus Transfection) transfection reagent Total RNA was extracted from cell lysates using the NucleoSpin RNA II kit (Macherey-Nagel) and eluted in water cDNA libraries were prepared from 100-200 ng RNA using a Illumina Stranded mRNA library Preparation Kit (Illumina, USA) following the manufacturer's protocol whereby adaptors are added to cDNA by A-tail mediated ligation. Index barcodes were added by PCR of 16 cycles. Unbound adaptors and indexes were eliminated by purification on AMPure magnetic beads (Beckman-Coulter). The resulting library featured an electrophoretic profile of 200-1000 bp, with a major peak of 375 bp, as visualized on a 3500 Fragment Analyzer (Agilent). A NextSeq 2000 sequencing system and a P4 50c flowcell (Illumina) were used to obtain 67-nt single-end double-indexed reads