title: Ion Torrent S5 XL sequencing; Proteogenomics uncovers critical elements of host response in bovine soft palate epithelial cells following in vitro infection with foot-and-mouth disease virus (FMDV)
design: Proteogenomics uncovers critical elements of host response in bovine soft palate epithelial cells following in vitro infection with foot-and-mouth disease virus (FMDV)
library_construction_protocol: Multilayers of bovine dorsal soft palate cells were propagated at the air-liquid interface for 5 weeks before infection. At days 0, 1 and 28, cells from 2 inserts were lysed with 750 μl of TRIzol Reagent (Life Technologies, Carlsbad, CA, USA) and frozen at -80 °C. 150 µL of trichlormethane (Carl Roth, Karlsruhe, Germany) was added to lysed cells in 750 μl TRIzol Reagent and the RNA-containing aqueous phase was then mixed with an equal amount of 100% ethanol (Carl Roth). Total RNA was extracted using the RNeasy Mini Kit (Qiagen, Hilden, Germany) with on-column DNase digestion with the RNase-Free DNase Set (Qiagen). Polyadenylated mRNA was isolated from 1-3 µg of high-quality total RNA using the Dynabeads mRNA DIRECT Micro kit (Invitrogen). For preparation of whole-transcriptome libraries the Ion Total RNA-Seq Kit v2 (Life Technologies) was used (1 - 100 ng mRNA, RNase III treatment at 37°C for 10 min). Libraries were quantified using the KAPA Library Quantification Kit Ion Torrent (Kapa Biosystems, Wilmington, MA, USA) on a CFX96 Real-Time PCR Detection System (Bio-Rad Laboratories, München, Germany).