title: Sample 24
organism: Homo sapiens
description: Protocols: The SARS-CoV-2 strain used in this study was isolated from a patient enrolled in a French clinical cohort of COVID-19 patients (ClinicalTrials.gov identifier NCT04262921; The sequence is available in the GISAID EpiCoV™ database under the accession BetaCoV/France/IDF0571/2020 (EPI_ISL_411218). Viral stocks were amplified and titrated by determination of the 50% tissue culture infectious dose (TCID₅₀/mL) on Vero cells (ATCC CRL-1586). The influenza A/Lyon/969/2009 (H1N1) strain was propagated and titrated on MDCK cells (ATCC CCL-34). A recombinant RSV A2 strain expressing mCherry (RSV-mCherry, kindly provided by Jean-François Eléouët, INRAE, Jouy-en-Josas, France) was propagated and quantified on LLC-MK2 cells (ATCC CCL-7). For infection, HAE were gently washed twice at the apical surface with Opti-MEM (Life Technologies) and subsequently mock-infected (Opti-MEM) or infected with SARS-CoV-2, IAV, or RSV at multiplicities of infection (MOI) of 0.01, 0.1, and 0.2, respectively. At defined time points, apical washes and basal media were collected and stored at −80°C for further analyses. Cells were lysed for total RNA extraction, which was used for RT-qPCR and RNA-seq. All experiments involving infectious SARS-CoV-2 material were performed in a biosafety level 3 (BSL-3) facility, while infections involving only IAV and/or RSV were conducted in a biosafety level 2 (BSL-2) laboratory. Briefly, HAE were lysed in RLT buffer (Qiagen RNeasy® Plus Mini Kit, ref. 74136) and scraped before collection. Total RNA was extracted using the same kit according to the manufacturer's instructions. RNA library preparation was realized following the manufacturer's recommendations (QuantSeq 3' mRNA-Seq V2 Library Prep Kit from LEXOGEN).
attributes.tag: replicate
attributes.value: replicate c
attributes.tag: disease
attributes.value: infectious disease
attributes.tag: organism
attributes.value: Homo sapiens
attributes.tag: organism
attributes.value: Homo sapiens
attributes.tag: collection date
attributes.value: not collected
attributes.tag: scientific_name
attributes.value: Homo sapiens
attributes.tag: common name
attributes.value: human
attributes.tag: organism part
attributes.value: lung epithelium
attributes.tag: developmental stage
attributes.value: adult
attributes.tag: isolate
attributes.value: not applicable
attributes.tag: geographic location (country and/or sea)
attributes.value: not collected
attributes.tag: infect
attributes.value: Simultaneous infection influenza H1N1/RSV