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Sample GSM4973648 Query DataSets for GSM4973648
Status Public on May 06, 2021
Title 15071_Het_PIC2
Sample type SRA
 
Source name immortalized epidermal keratinocytes from ear punch
Organism Canis lupus
Characteristics cbd103 genotype: yB
estimated age: 3-4 years
cell culture treatment: polyI:C
cell culture experiment: polyI:C experiment_immortalized
Treatment protocol polyI:C experiment: Keratinocytes were plated at 1.5E4 cells per well of a 12-well plate with 3T3 feeder cells in FAD medium. When cells were at ca. 30% confluence, they were switched to a low-serum medium (FAD + 0.5% BCS) without RI and incubated for 24 hours. Cells were then treated with 1 ug/ml polyI:C (Sigma-Aldrich) or vehicle control (sterile, endotoxin-free water) for 24 hours. CDV experiment: Keratinocytes were plated in duplicate on 24-well plates at 8E4 cells per well in FAD + 5% BCS + RI, without feeder cells. After a 24 hour incubation, the medium was changed to FAD + 0.5% BCS. After an additional 24 hours, cells were infected with CDV at an MOI of 100 TCID50/ml (or were provided medium in same volume, as control). Cells were collected 5 days post-infection.
Growth protocol Primary wolf epithelial keratinocytes were grown with 3T3 feeder cells in FAD medium: 1:1 DMEM:F12 base media [ThermoFisher Scientific] + 5% BCS + 0.4 ug/ml hydrocortisone + 10 ng/ml epidermal growth factor (EGF) + 1% Penicillin-Streptomycin [ThermoFisher Scientific] + 10 uM ROCK inhibitor Y-27632 [Cayman Chemical, Ann Arbor, MI, USA].
Extracted molecule polyA RNA
Extraction protocol Trizol Plus RNA Purification Kit with DNAse treatment (RNAse-Free DNAse Set, Ambion) and column cleanup (PureLink RNA Mini Kit, Invitrogen)
Half-reactions of the TruSeq RNA Sample Preparation Kit (Illumina)
 
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina HiSeq 4000
 
Data processing Reads were trimmed with Trim Galore (Krueger, 2015) to remove adapter sequence and base pairs with Phred score < 20 from the ends of reads, keeping only reads ≥ 20 basepairs long after trimming.
Trimmed reads were mapped to the dog genome (canfam3.1) using two-pass mapping with STAR (Dobin et al., 2013). Only uniquely mapped reads were retained.
Gene expression was quantified using HT-Seq (Anders et al., 2014) with the “union” mode using the Canis_familiaris.CanFam3.1.94.gtf file from Ensembl.
Genome_build: canfam3.1
Supplementary_files_format_and_content: allwolfcounts.txt is a tab delimited text file showing the output from HTseq, which provides the number of reads mapping to each gene for each RNA-Seq sample/library.
 
Submission date Dec 14, 2020
Last update date May 06, 2021
Contact name Rachel A Johnston
E-mail(s) racheljohnston7@gmail.com
Organization name Duke University
Department Evolutionary Anthropology
Lab Jenny Tung
Street address 130 Science Dr, Rm 108 Biological Sciences
City Durham
State/province NC
ZIP/Postal code 27708
Country USA
 
Platform ID GPL26727
Series (1)
GSE163163 Gene expression response of wolf epidermal keratinocytes to PolyI:C and canine distemper virus
Relations
BioSample SAMN17077973
SRA SRX9683825

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record

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