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| Status |
Public on Mar 12, 2017 |
| Title |
Gray_Boy |
| Sample type |
SRA |
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| Source name |
Feline adipose-derived MSCs cultured in standard tissue culture medium
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| Organism |
Felis catus |
| Characteristics |
cell type: feline primary adipose-derived MSC culture
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| Treatment protocol |
Untreated adipose-derived MSC cultures were utilized in this study. Once cultures reached the desired density during standard passaging, the cells were harvested for isolation of toal RNA.
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| Growth protocol |
The MSC cultures were cultured in standard Minimum Essential Medium α [MEMα] tissue culture medium supplemented with 10% fetal bovine serum, 1% L-glutamine, and 1% penicillin/streptomycin. MSCs from passages 3–4 were used for experimentation.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated from primary ASC cultures using the RNeasy Mini Kit (Qiagen, Inc.), RNA quantity and quality were assessed on a NanoDrop spectrophotometer (Thermo Scientific) and the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA), respectively.
Transcriptome profiling was performed using a directional, strand-specific mRNA-Seq approach. Briefly, indexed RNA-Seq libraries were prepared from 200 ng total RNA using the KAPA Stranded mRNA-Seq Kit (Kapa Biosystems, Inc.) according to the manufacturer’s standard protocol. Poly-adenylated mRNA was purified from total RNA and ribosomal RNA removed by binding to oligo(dT) beads, which was followed by RNA fragmentation by incubation at 94ºC in the presence of magnesium. Double-stranded cDNA was then generated by random-primed first-strand synthesis and subsequent second strand synthesis in the presence of dUTP for strand marking. The double-stranded cDNA was then 3’-A tailed and indexed, Illumina-compatible adapters were ligated. The libraries were then enriched by high-fidelity PCR amplification (15 cycles) with KAPA HiFi HotStart DNA Polymerase and adapter-specific primers.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 4000 |
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| Description |
Illimuna next-generation sequencing (paired-read)
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| Data processing |
Image processing, base calling, quality scoring (Phred), and sample demultiplexing were executed by HiSeq Control Software with Real Time Analysis (HCS v3.3.41/RTA 2.5.2) and bcl2fastq Conversion Software (Illumina; San Diego, CA)
FASTQ-formatted sequence data was analyzed using a standard HISAT (hierarchical indexing for spliced alignment of transcripts)-Cufflinks workflow. RNA-Seq sequence reads (FASTQ format) were aligned to the reference cat genome assembly (Nov. 2014, ICGSC Felis Catus 8.0) using HISAT software. Gene- and transcript-level expression were comprehensively quantified with Cufflinks software, which performed 1) transcript assembly, 2) identification of splice variants, 3) quantification of expression as FPKM (fragments per kilobase of transcript per million mapped reads) values, and 4) normalization. Normalized FPKM values (Cuffnorm output) were utilized for downstream analysis steps.
Processed data are Cufflinks-Cuffnorm output files and contain Gene_Symbol (Ensembl ID), FPKM values, and FPKM status.
Genome_build: Nov. 2014, ICGSC Felis Catus 8.0
Supplementary_files_format_and_content: Tab-delimited text files include Ensembl ID, FPKM values, and FPKM status for each transcript and were prepared from the Cufflinks-Cuffnorm gene-level output files.
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| Submission date |
Feb 10, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Clifford G. Tepper |
| E-mail(s) |
cgtepper@ucdavis.edu
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| Phone |
916-734-7195
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| Organization name |
UC Davis School of Medicine
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| Department |
Biochemistry and Molecular Medicine
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| Street address |
4645 2nd Avenue, Room 2300A
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| City |
Sacramento |
| State/province |
CA |
| ZIP/Postal code |
95817 |
| Country |
USA |
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| Platform ID |
GPL23056 |
| Series (1) |
| GSE94773 |
Human and feline adipose-derived mesenchymal stem cells have comparable immunomodulatory functions |
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| Relations |
| BioSample |
SAMN06319451 |
| SRA |
SRX2549941 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2482745_Gray_Boy_cuffnorm_genes.txt.gz |
123.3 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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