 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Oct 23, 2019 |
| Title |
HODay3replicate2 |
| Sample type |
SRA |
| |
|
| Source name |
Tenotomy site of a burn/tenotomy mouse
|
| Organism |
Mus musculus |
| Characteristics |
treatment: A partial-thickness scald burn injury was administered to animals
timepoint: cells recruited to injury site at day 3
|
| Treatment protocol |
A partial-thickness scald burn injury was administered to animals according to a previously described protocol by a single surgeon.3,57 Briefly, mice were anesthetized with inhaled isoflurane. Dorsal hair was closely clipped, and an aluminum block heated to 60°C was exposed to the dorsal region over 30% of the total body surface area for 18 s to achieve a partial-thickness burn injury. Each mouse then received a concurrent sterile dorsal hind limb tendon transection at the midpoint of the Achilles tendon (Achilles tenotomy) with placement of a single 5-0 vicryl suture to close the skin. Pain management was achieved with subcutaneous injections of buprenorphine (Buprenex, Reckitt Benckiser Pharmaceuticals) every 12 hr for 2 days.
Post injury harvested tissue samples were digested for 45 minutes in 0.3% Type 1 Collagenase and 0.4% Dispase II (Gibco) in Roswell Park Memorial Institute (RPMI) medium at 37°C under constant agitation at 120rpm. Digestions were subsequently quenched with 10% FBS RPMI and filtered through 40μm sterile strainers. Cells were then washed in PBS with 0.04% BSA, counted and resuspended at a concentration of ~1000 cells/ul. Cell viability was assessed with Trypan blue exclusion on a Countess II (Thermo Fisher Scientific) automated counter and only samples with >85% viability were processed for further sequencing.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Single-cell 3’ library generation was performed on the 10x Genomics Chromium Controller following the manufacturers protocol for the v2 reagent kit (10X Genomics, Pleasanton, CA, USA). Cell suspensions were loaded onto a Chromium Single-Cell A chip along with reverse transcription (RT) master mix and single cell 3’ gel beads, aiming for 2000-6000 cells per channel. In this experiment, 8700 cells were encapsulated into emulsion droplets at a concentration of 700-1200 cells/ul which targets 5000 single cells with an expected multiplet rate of 3.9%. Following generation of single-cell gel bead-in-emulsions (GEMs), reverse transcription was performed and the resulting Post GEM-RT product was cleaned up using DynaBeads MyOne Silane beads (ThermoFisher Scientific, Waltham, MA, USA). The cDNA was amplified, SPRIselect (Beckman Coulter, Brea, CA, USA) cleaned and quantified then enzymatically fragmented and size selected using SPRIselect beads to optimize the cDNA amplicon size prior to library construction. An additional round of double-sided SPRI bead cleanup is performed after end repair and A-tailing. Another single-sided cleanup is done after adapter ligation. Indexes were added during PCR amplification and a final double-sided SPRI cleanup was performed. Libraries were quantified by Kapa qPCR for Illumina Adapters (Roche) and size was determined by Agilent tapestation D1000 tapes. Read 1 primer sequence are added to the molecules during GEM incubation. P5, P7 and sample index and read 2 primer sequence are added during library construction via end repair, A-tailing, adaptor ligation and PCR. Libraries were generated with unique sample indices (SI) for each sample.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 4000 |
| |
|
| Description |
HO model (30% body surface area dorsal burn and Achilles tenotomy) was used in C57 and LysmCre/Tgfb1fx/fx mice
Day 3
HO_Day3_Markers
|
| Data processing |
Cell Ranger Single Cell Software Suite 1.3 was used to perform sample de-multiplexing, barcode processing, and single cell gene counting (Alignment, Barcoding and UMI Count) at the University of Michigan Biomedical Core Facilities DNA Sequencing Core.
Seurat R package was used to perform unsupervised clustering and identification of cell-type specific markers.
Genome_build: GRCh38
Supplementary_files_format_and_content: barcodes.tsv, genes.tsv, and matrix.mtx files.
|
| |
|
| Submission date |
Feb 04, 2019 |
| Last update date |
Oct 25, 2019 |
| Contact name |
Rajasree Menon |
| E-mail(s) |
rajmenon@umich.edu
|
| Phone |
7346159720
|
| Organization name |
University of Michigan
|
| Department |
Department of Computational Medicine and Bioinformatics
|
| Street address |
4544E, MSRB2, Catherine Street, University of Michigan
|
| City |
Ann Arbor |
| State/province |
Michigan |
| ZIP/Postal code |
48109 |
| Country |
USA |
| |
|
| Platform ID |
GPL21103 |
| Series (1) |
| GSE126060 |
Role of myeloid cells in heterotopic ossification (HO) in a burn and incision-induced mouse model |
|
| Relations |
| BioSample |
SAMN10869537 |
| SRA |
SRX5330774 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3589979_HODay3replicate2_barcodes.tsv.gz |
9.6 Kb |
(ftp)(http) |
TSV |
| GSM3589979_HODay3replicate2_genes.tsv.gz |
212.7 Kb |
(ftp)(http) |
TSV |
| GSM3589979_HODay3replicate2_matrix.mtx.gz |
20.7 Mb |
(ftp)(http) |
MTX |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
|
|
|
|
 |
