P3 flow cell

For RNA-Seq analysis, three independent replicates were prepared for each treatment group, Mock, MOI 0.01-, and MOI 0.1-exposed HBMEC cultures, after 6 and 24 h. Total RNA was isolated via the miRNeasy micro kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. The RNA was quantified by O.D. measurement before being assessed for quality by chip-based capillary electrophoresis using Agilent 2100 Bioanalyzer RNA 6000 Pico assays (Agilent Technologies, St Clara, CA, USA; Part # 5067-1513).
Libraries were prepared from 150 nanograms (ng) of DNA-free total RNA using the Universal Plus mRNA-Seq Library Prep Kit (NuGEN Technologies, Inc., San Francisco, CA, USA; Part # 0508-96). The quality and size distribution of the amplified libraries was determined by chip-based capillary electrophoresis on Agilent 2100 Bioanalyzer High Sensitivity DNA assays (Agilent Technologies; Part # 5067-4626). Libraries were quantified using the Takara Library Quantification Kit (Shiga, Japan; Part # 638324). The libraries were pooled at equimolar concentrations and diluted prior to loading onto a P3 flow cell (Illumina, San Diego, CA, USA; Part # 20027800) with the P3 300 Cycle reagent kit (Illumina, San Diego, CA; Part # 20038732) on the NextSeq2000 instrument (Illumina, San Diego, CA, USA).

For RNAseq analysis, three independent replicates were prepared for each treatment groups: Mock, MOI 0.01- and MOI 0.1-infected cultures, at 6 and 24 hpi. Total RNA was isolated using the miRNeasy micro kit (Qiagen) according to the manufacturer's instructions. The RNA was quantified by O.D. measurement before being assessed for quality by chip-based capillary electrophoresis using Agilent 2100 Bioanalyzer RNA 6000 Pico assays (Agilent Technologies; Part # 5067 – 1513).
Libraries were prepared from 150 nanograms (ng) of DNA-free total RNA using the Universal Plus mRNA-Seq Library Prep Kit (NuGEN Technologies, Inc.; Part # 0508 – 96). The quality and size distribution of the amplified libraries were determined by chip-based capillary electrophoresis on Agilent 2100 Bioanalyzer High Sensitivity DNA assays (Agilent Technologies; Part # 5067 – 4626). Libraries were quantified using the Takara Library Quantification Kit (Part # 638324). The libraries were pooled at equimolar concentrations and diluted prior to loading onto a P3 flow-cell (Illumina; Part # 20027800) with the P3 300 Cycle reagent kit (Illumina; Part # 20038732) on the NextSeq2000 instrument.