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| Status |
Public on Dec 08, 2022 |
| Title |
scRNA-Seq_Day7_Back_RNA |
| Sample type |
SRA |
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| Source name |
Day7_Back_Wound
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| Organism |
Rangifer tarandus |
| Characteristics |
tissue: back skin
time: day 7
facs strategy: Dead cells excluded using the forward/side scatter gating and viability dye (Fixable Viability Dye eFluor™ 780)
library preparation: 10X Genomics Single Cell RNAseq v3
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| Treatment protocol |
Animals were anesthetized using injectable anesthesia (Medetomidine, 0.07-0.15mg/kg IM with Azaperone, 0.2mg/kg IM if additional duration of anesthesia required). The animals were blindfolded, their eyes lubricated (Optixcare eye lube), and nasal insufflation of pure oxygen was administered throughout the procedure. Monitoring of anesthetic depth, hear and respiratory rate and temperature were monitored by a licenced animal health technologist with expertise in wildlife handling and anesthesia.
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| Growth protocol |
All Reindeer (Rangifer tarandus) used for this project were housed in an outdoor enclosure at the University of Calgary Veterinary Sciences Research station in Calgary, Alberta for the entire length of the procedure. All experiments were done in accordance with guidelines set out by the University Animal Welfare Committee (UAWC), the Animal Care Committee (ACC), and with the recommendations and regulations of the Canadian Council on Animal Care and the Province of Alberta Animal Protection Act and Regulation.
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
Following the collection of 12mm back and velvet skin excisions, the samples were minced and incubated in dispase (StemCell Technologies) for 10-15 minutes at 37°C to remove epidermis. Remaining dermis tissue was minced and digested using collagenase (0.2%, Sigma) in 37 degree C water bath for 1-3 hours. To further dissociate the tissue and liberate single cells, the gentleMACS™ Octo Dissociator (Miltenyi Biotec) was used for 6 minutes on a homogenization program. DNase I (1x - Sigma) was also added to the tissue for the last 30 minutes in the water bath. Single cell suspensions were resuspended in cold FACS staining buffer (1% bovine serum albumin in Hank’s Buffered Salt Solution, HBSS; Life Technologies), strained through 100 µm and 70 µm filters (Falcon) and centrifuged at 1200 rpm for 6 minutes. Cell pellets were resuspended in FACS staining buffer and quantified. Viability dye eFlour780 (eBiosciences) was used to exclude dead cells using FACSAria III (BD Biosciences).
All samples were prepared according to 10X Genomics ChromiumTM Single Cell 3’ Reagent Guidelines v3 Chemistry. Briefly, single cells were sorted into 0.1% BSA–HBSS and partitioned into Gel Bead-In-EMulsions (GEMs) using 10xTM GemCodeTM Technology. This process lysed cells and enabled barcoded reverse transcription of RNA, generating full-length cDNA from poly-adenylated mRNA. DynaBeads® MyOneTM Silane magnetic beads were used to remove leftover biochemical reagents, then cDNA was amplified by PCR over 10 cycles. Quality control size gating was used to select cDNA amplicon size prior to library construction. Read 1 primer sequences were added to cDNA during GEM incubation. P5 primers, P7 primers, i7 sample index, and Read 2 primer sequences were added during library construction. Quality control and cDNA quantification was performed using Agilent High Sensitivity DNA Kit. For all samples, sequencing was performed using a combination of Illumina NovaSeq SP and S2 Flow cells to ensure all samples received 25,000 reads/cell at minimum.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
FACS sorted cells: 50,000 cells; Recovered: 4,112 cells
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| Data processing |
All raw FASTQs reads were aligned to bovine (Bos taurus) genome using the STAR algorithm. Samples 1-8 were quantified through the 10X Genomics Cellranger v3 single cell pipeline, with default and recommended parameter. Only mapped reads were used for normalization before aggregating cells. The resulting gene-barcode matrix was imported into Seurat R toolkit for quality control and all downstream analysis. Back and antler samples were integrated for each time point using Canonical Correlation Analysis (CCA) and Mutual Nearest Neighbor (MNM) techniques employed by Seurat v3.1.1.
Genome_build: Bos_taurus (Downloaded from Ensembl: https://uswest.ensembl.org/Bos_taurus/Info/Index Made cellranger compatible using cellranger-mkref pipeline; Reference Genome GTF file is available on series record.
Supplementary_files_format_and_content: Filtered gene - barcode matrix output from CellRanger-count (transcriptome = Bos_taurus)
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| Submission date |
Jan 02, 2020 |
| Last update date |
Dec 08, 2022 |
| Contact name |
Jeff Biernaskie |
| E-mail(s) |
jabierna@ucalgary.ca
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| Phone |
4032107306
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| Organization name |
University of Calgary
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| Department |
Comparative Biology and Experimental Medicine
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| Lab |
Biernaskie Lab
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| Street address |
3330 Hospital Drive NW
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| City |
Calgary |
| State/province |
Alberta |
| ZIP/Postal code |
T2N4N1 |
| Country |
Canada |
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| Platform ID |
GPL27966 |
| Series (2) |
| GSE142854 |
Single-cell RNA-Seq datasets supporting fibroblast polarization drives skin regeneration versus fibrosis in adult reindeer |
| GSE168748 |
Skin regeneration is enabled in the absence of fibroblast inflammatory priming |
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| Relations |
| BioSample |
SAMN13713557 |
| SRA |
SRX7493859 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4244983_Sample_6_Day7_Back_RNA_barcodes.tsv.gz |
21.6 Kb |
(ftp)(http) |
TSV |
| GSM4244983_Sample_6_Day7_Back_RNA_features.tsv.gz |
187.3 Kb |
(ftp)(http) |
TSV |
| GSM4244983_Sample_6_Day7_Back_RNA_matrix.mtx.gz |
19.7 Mb |
(ftp)(http) |
MTX |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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