|
| Status |
Public on May 06, 2021 |
| Title |
15054P |
| Sample type |
SRA |
| |
|
| Source name |
primary epidermal keratinocytes from ear punch
|
| Organism |
Canis lupus |
| Characteristics |
cbd103 genotype: yy
estimated age: 4-5 years
cell culture treatment: polyI:C
cell culture experiment: polyI:C experiment
|
| 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 |
SAMN17077901 |
| SRA |
SRX9683773 |
