Nd 2000 spectrophotometer

The expression of genes (Table 1) related to the HA receptor family (CD44, ICAM, and HMMR/RHAMM), tight junction protein (ZO1), GAG synthesis enzymes (CSGALNACT1 and CSGALNACT2), markers of pain and inflammation (COX1, NGF) and tissue remodeling (TIMP1 and MMP1) were assayed. Total RNA was isolated using RNeasy Mini Kit (Qiagen, Crawley, United Kingdom) and quantity assessed by nanodrop (ND-2000 Spectrophotometer (ThermoFisher Scientific, United Kingdom). The integrity of RNA samples was assessed using a bioanalyser (Agilent, Dublin, Ireland). Samples with a 260:280 nm ratio of 1.8–2.1 for purity and an RNA integrity number greater than 8.5 were used in subsequent experiments. Isolated RNA was stored at −80 °C before transformation into cDNA using the cDNA synthesis first strand synthesis kit (Roche Life Sciences, Ireland) all cDNA stored at −20 °C. All reactions and controls were performed in triplicate using real time ready custom 384-well plates on a Lightcycler 480 system (Roche Life Sciences, Ireland). Thermal cycling conditions were as recommended by the manufacturer (Applied Biosystems, Paisley, United Kingdom). Relative changes in gene expression were determined using the ΔΔ^CT method.

Total RNA was isolated using Trizol reagent (Invitrogen) according to the manufacturer's instructions. RNA quality and quantity were determined by ND-2000 Spectrophotometer (Thermo).

Total RNA was isolated using QIAzol lysis reagent (79306, Qiagen, Germany). RNA quality was assessed by Agilent 2100 bioanalyzer (Agilent Technologies, Amstelveen, The Netherlands), and RNA quantification was performed using an ND‐2000 Spectrophotometer (Thermo Fisher, DE, USA).

Total RNA was isolated using the Trizol reagent (Invitrogen, Waltham, MA, United States). RNA quality was assessed with the Agilent 2100 bioanalyzer using the RNA 6000 Nano Chip (Agilent, Santa Clara, CA, United States), and RNA quantification was performed using an ND-2000 Spectrophotometer (Thermofisher, Waltham, MA, United States).

The total RNA of Calu-3 cells infected with or without 0.004 MOI MERS-CoV or treated for 24 h with MERS-CoV, and 10 μM of the indicated compounds was isolated using RNeasy Mini Kits (Qiagen, Valencia, CA, USA). RNA quality was assessed using the Agilent 2100 bioanalyzer with the RNA 6000 Nano Chip (Agilent Technologies, Amstelveen, The Netherlands), and RNA was quantified using an ND-2000 Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). A library was constructed using QuantSeq 3′ mRNA-Seq Library Prep Kits (Lexogen GmbH, Vienna, Austria). High-throughput sequencing was performed as single-end 75 sequencing using NextSeq 500 (Illumina, Inc., San Diego, CA, USA). QuantSeq 3′ mRNA-seq reads were aligned using Bowtie2 [13 (link)]. DEGs were determined based on counts from unique and multiple alignments using coverage in BEDTools [14 (link)]. The read count (RC) data were processed based on a quantile normalization method using EdgeR within R [15 ] using Bioconductor [16 (link)]. For DEGs, Gene Ontology (GO) analyses [17 (link)] were performed using clusterProfiler (Version 3.18.1) [18 (link)] in R (Version 4.0.3), which supports the statistical analysis and visualization of functional profiles for genes and gene clusters.