2025-03-01
ENA first public
ERC000011
ENA-CHECKLIST
European Institute of Oncology
INSDC center name
public
INSDC status
ERS22881966
SRA accession
E-MTAB-14745:Sample 2
Submitter Id
ArrayExpress
broker name
primary cell line
cell_line
astrocyte
cell_type
not collected
collection date
human
common name
Protocols: Astrocytes with 90% confluency were used for infection at a MOI of 0.2. Cell were incubated either with Delta (BA15) or Omicron (BA42) or PBS (phosphate buffer saline, pH 7.2, negative control) for one hour. After incubation, medium was aspirated, the cells were washed once with PBS and fresh complete astrocyte medium was added. The cells were then allowed to culture for 6 h at 37 °C under 5% CO2. RNA was isolated from the cells.  Culture of astrocytes  Human astrocytes (primary cells, ScienCell) were cultured in 6-well plates coated with poly-L-lysine (ScienCell). The seeding density was 100,000 per well (fourth passage). Cells were cultured in astrocyte medium supplemented with astrocyte growth supplement (ScienCell), penicillin (10,000 units/ml), streptomycin (10,000 μg/ml), and 1% FBS (ScienCell) at 37 °C under 5% CO2 atmosphere until 90% confluence (approximately 300,000 cells/well).  Viruses The viruses were derived from a nasopharyngeal swab and gargle sample of COVID-19 patients from Slovakia. Both isolates were deposited in the European virus archive GLOBAL EVAg). The strain Slovakia/SK-BMC-BA15/2021 (available at https://www.european-virus-archive.com/virus/sars-cov-2-strain-slovakiask-bmc-ba152021-b16172-variant-aka-delta-voc-or-indian-variant) was isolated in June 2021 and belongs to the lineage B.1.617.2 (Delta). The strain Slovakia/SK-BMC-BA42/2022 was isolated in July 2022 (https://www.european-virus-archive.com/virus/sars-cov-2-strain-slovakiask-bmc-ba422022-omicron-voc-ba52) and belongs to the BA.5.2 lineage (Omicron VOC). The identity of the viruses was confirmed by sequencing. The complete genome sequences are available in EVAg portal.  Virus cultivation and titer determination One milliliter of each clinical sample was propagated in Vero E6 cells cultured in 25 cm2 flasks in Dulbecco's modified essential medium containing 5% FBS  (Sigma Aldrich), penicillin (10,000 units/ml), streptomycin (10,000 μg/ml), 2 mM L-glutamine and incubated at 37°C with 5% CO2 for 1 hour. After one hour the medium was removed, cells were washed with PBS and new medium was added. The cells were further cultured until 80% of visible cytopathic effect. Then cell culture was repeatedly frozen and thawed, the cell supernatant was centrifuged at 4400 rpm for 10 min. 1 ml of infectious cell supernatant was passaged and propagated in Vero E6 cells one more time to obtain a working stock. The rest of supernatant was aliquoted and stored at -80 °C. Viral titre was determined by standard plaque assay in Vero E6 cells.    Astrocytes with 90% confluency were used for infection at a MOI of 0.2. Cell were incubated either with Delta (BA15) or Omicron (BA42) or PBS (phosphate buffer saline, pH 7.2, negative control) for one hour. After incubation, medium was aspirated, the cells were washed once with PBS and fresh complete astrocyte medium was added. The cells were then allowed to culture for 6 h at 37 °C under 5% CO2.  After incubation of cells (described above) the medium was removed, the cells were washed with PBS, and lysis buffer (RLT, Qiagen, Germany) was added. RNAs were isolated with the RNeasy Mini Kit (Qiagen) and treated with DNase I (Qiagen) according to the manufacturer's instructions. RNA concentrations were measured by nanodrop (Thermo Fisher Scientific) and integrity was monitored by capillary electrophoresis (Fragment analyzer, Advanced Analytical Technologies, Inc., USA). RNA was stored at -80°C and used for preparation of libraries for RNA sequencing.  Libraries for RNA sequencing were prepared using QuantSeq 3′ mRNA-Seq Library Prep Kit (Lexogen, Austria) exactly as described in protocol provided with kit.
description
normal
disease
wild type genotype
genotype
not collected
geographic location (country and/or sea)
infection with SARS-CoV-2 Omicron [Slovakia/SK-BMC-BA42/2022]
infect
not applicable
isolate
Homo sapiens
organism
Homo sapiens
organism
brain
organism part
Homo sapiens
scientific_name
Sample 2
title