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Sample GSM5555592

Query DataSets for GSM5555592

Status

Public on Aug 31, 2022

Title

DH82_H_d3_2

Sample type

SRA

Source name

DH82_cells

Organism

Canis lupus familiaris

Characteristics

cell line: DH82_cells
infection: control
stress: hypoxia
time: day 3

Treatment protocol

DH82 cells persistently infected with canine distemper virus strain Onderstepoort (DH82Ondpi) were seeded at a density of 0.33*106 cells / T25 flask. Cells were cultured at normoxia, 5% CO2, 37°C, humidified atmosphere, in minimal essential medium (MEM) with Earle’s salts (PAA, Cölbe, Germany) supplemented with 10% fetal calf serum (PAA) , 1% penicillin/streptomycin (PAA) and 1% non-essential amino acids (Sigma-Aldrich, Taufkirchen, Germany). 3 days after seeding a medium change with the aforementioned medium was performed. 6 days after seeding the medium was changed according to the planned culture conditions. Control cultures received the aforementioned medium again and were cultured at 37°C, normoxia, 5% CO2, in a humidified atmosphere as described above. For hypoxic conditions, cells received the aforementioned medium again and were cultured at 37°C, 1% O2, 5% CO2, in a humidified atmosphere. For starvation conditions, cells received the aforementioned medium without fetal calf serum and were cultured at 37°C, normoxia, 5% CO2, in a humidified atmosphere.

Growth protocol

DH82 cells were obtained from the European Collection of Authenticated Cell Cultures (ECACC).

Extracted molecule

total RNA

Extraction protocol

Cells were harvested 1 d and 3 days after treatment and frozen at a density of 3*106 cells / aliquot. RNA isolation was performed directly out of cryopreserved cells. Cells were isolated using TRIzol® (Invitrogen / ThermoFisher, Langenselbold, Germany) according to manufacturer’s instructions. After isolation RNA was cleaned up using RNeasy Mini-Kits (Qiagen, Hilden, Germany) with on column DNase treatment (RNase free DNase, Qiagen).
100ng total RNA was used for preparation of RNA Sequencing library with NEBNext® Single Cell/Low Input RNA Library Prep Kit for Illumina® (New England Biolabs).
The libraries were sequenced on Illumina NovaSeq 6000 using NovaSeq 6000 S1 Reagent Kit (300 cycles, paired end run 2x 150 bp) with an average of 6 x10E7 reads per RNA sample

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NovaSeq 6000

Data processing

Reads were quality checked with package FastQC (version 0.11.4,), then trimmed using Trimgalore (version 0.4.4) with default settings.
Trimmed reads were mapped to Canis_lupus_familiaris genome using STAR aligner (Dobin and Gingeras, 2015) with default settings.
Mapped reads were counted using RsubRead (version 1.32.4, (Liao et al., 2019)). Raw counts were then normalized using DESeq2 (version 1.16.1, (Love et al., 2014)).
Genome_build: ENSEMBL release-100/Canis_lupus_familiaris.CanFam3.1.dna and CDV genome sequence (proprietry sequence, 99% identity to published GenBank Accession Number AF378705.1).
Supplementary_files_format_and_content: txt matrix with log2 normalized values per sample.

Submission date

Sep 02, 2021

Last update date

Aug 31, 2022

Contact name

Klaus Schughart

E-mail(s)

labschughart@online.de

Phone

+49-159-0483-7911

Organization name

University Münster