2025-10-30
ENA first public
ERC000011
ENA-CHECKLIST
Friedrich-Loeffler-Institut, Institute of Diagnostic Virology
INSDC center name
public
INSDC status
ERS23771350
SRA accession
E-MTAB-14894:0816_24hpi_R1_infected_early
Submitter Id
ArrayExpress
broker name
not collected
collection date
common carp
common name
Protocols: Cell pellets were collected in ATV after removal of supernatants. Cells were cultured in 6-well plates (Corning) at a seeding density of approximately 1 × 10⁶ cells per well in 5 ml medium supplemented with 10% FBS. They were maintained at 26°C in a humidified environment with 5% CO₂.  Cell pellets were collected from two independent experiments to analyse both early and late infection stages. For the early stage experiment, samples were collected at 4, 8, 24 and 48 hours post infection (hpi). In the late stage experiment, samples were collected at 72, 96, 120 and 240 hpi.  Negative controls were treated with 500 µl of the same medium used for GSB cultivation, replacing 500 µl of the culture medium. Control cell pellets were collected at 7 hours post attachment (hpi) and at 0, 4, 8, 24 and 48 hpi for the early stage. For the late stage, control samples were collected at 7 hpa and at 0, 72, 96, 120 and 240 hpi. WhSBV infection was performed at a 1:10 ratio by replacing 500 µl of culture medium with 500 µl of WhSBV inoculum. Cell pellets were lysed in 1 ml TRIzol LS reagent (Life Technologies) and shaken vigorously for 10 minutes at room temperature. After the addition of 200 µl chloroform (Carl Roth GmbH & Co. KG) and centrifugation at 13,000 × g for 10 minutes at 4°C, 200 µl of the upper aqueous phase was collected for RNA extraction. Total RNA was isolated using the Agencourt RNAdvance Tissue Kit (Beckman Coulter) with the KingFisher Flex Purification System (Thermo Fisher Scientific) according to the manufacturer's protocol. DNase I digestion (Qiagen) was performed prior to final elution to remove residual genomic DNA. The amount of RNA was measured using a NanoDrop Lite spectrophotometer (Thermo Fisher Scientific). Poly(A) RNA was then purified using the Dynabeads mRNA DIRECT Purification Kit (Invitrogen) according to the manufacturer's instructions. ERCC spike-in mix (Invitrogen) was used as internal control. Fragmentation and strand-specific construction of whole transcriptome libraries was performed using the Collibri Stranded RNA Library Prep Kit for Illumina Systems (Invitrogen). The quality and integrity of both the RNA and the final library were assessed using the Agilent TapeStation 4150 (Agilent Technologies) with the appropriate chips and reagents. The final libraries were quantified using the Qubit dsDNA HS Assay Kit (Invitrogen) in conjunction with the Qubit 2.0 Fluorometer (Invitrogen).
description
wild type genotype
genotype
not collected
geographic location (country and/or sea)
not applicable
isolate
Cyprinus carpio
organism
Cyprinus carpio
organism
brain
organism part
Cyprinus carpio
scientific_name
WhSBV infected
stimulus
CCB
strain
0816_24hpi_R1_infected_early
title