mRNA profiling in pooled human umbilical vein endothelial cells stimulated with Coronavirus Disease 2019 (COVID-19) patient plasma

The purpose of this study was to identify mRNAs that were dysregulated after exposure to COVID-19 patient plasma and thus possibly contribute to vascular inflammation. Therefore, we conducted a multi-center, retrospective longitudinal cohort study enrolling 142 patients with laboratory-confirmed SARS-CoV-2 infection who presented to two Canadian hospitals from May 2020 – December 2020 along with a cohort of 27 SARS-CoV-2 patients with mild upper respiratory tract symptoms and 69 SARS-CoV-2-negative patients from the ICU. Blood was biobanked from SARS-CoV-2 positive patients in the emergency department (mild), ward (moderate) or intensive care unit (severe). Assessment of gene regulatory networks, gene set enrichment analysis, and in vitro permeability follow-up suggested functional reductions in junctional protein expression. Following this, confirmed critical reductions in VE-cadherin and ZO-1 which may drive pathology in moderate and severe cases of COVID-19. Overall design: RNA was extracted from pooled human umbilical vein endothelial cells (pHUVECs) that have either been left unstimulated (PBS-/- control) or have been stimulated with 20% (v/v) plasma from either: (1) SARS-CoV-2 negative patients with mild upper respiratory tract symptoms, (2) patients with mild COVID-19, (3) patients with moderate COVID-19, or (4) patients with severe COVID-19. Sample Groups: (1) n=5 unstimulated (2) n=5 SARS-CoV-2 negative patients (mild respiratory tract symptoms) (3) n=5 patients with mild COVID-19 (4) n=5 patients with moderate COVID-19 (5) n=5 patients with severe COVID-19 Samples were co-incubated for six hours prior to the isolation of RNA using a Qiagen RNeasy Plus Micro Kit (Cat#: 74004) only after washing twice with ice-cold Dulbecco's Phosphate Buffered Saline (PBS) without magnesium and calcium. RNA quantities and quality were assessed using the Agilent 2100 Bioanalyzer with all samples passing a quality control threshold (RIN = 7.0) to proceed to library preparations and RNA-seq. A total amount of 20 ng RNA per sample was used as input material for the RNA sample preparations. Sequencing libraries were generated using NEBNext® UltraTM RNA Library Prep Kit for Illumina® following manufacturer's recommendations and index codes were added to attribute sequences to each sample. Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in NEBNext First Strand Synthesis Reaction Buffer (5X). First strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase (RNase H-). Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3' ends of DNA fragments, NEBNext Adaptor with hairpin loop structure were ligated to prepare for hybridization. In order to select cDNA fragments of preferentially 150~200 bp in length, the library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA). Then 3 µL USER Enzyme was used with size-selected, adaptor-ligated cDNA at 37 °C for 15 minutes followed by five minutes at 95 °C before PCR. PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. The resulting PCR products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system. Sequencing was carried out on an Illumina NovaSeq® 6000 (Illumina, San Diego, California, United States), using paired end 2×150 bp chemistry at a depth of 20 million reads per sample.