|
| Status |
Public on Sep 10, 2021 |
| Title |
mock-3 |
| Sample type |
SRA |
| |
|
| Source name |
Lymph node
|
| Organism |
Mus musculus |
| Characteristics |
tissue: Lymph node
strain: C57BL/6
genotype: WT
inoculation: mock
time: 24 hpi
|
| Treatment protocol |
WT C57BL/6 mice were mock-inoculated (n = 3) or inoculated with 10e3 PFU of CHIKV (n = 3) in the left rear footpad.
|
| Growth protocol |
Mice were housed and bred at the University of Colorado School of Medicine under specific pathogen-free conditions and were distributed randomly into groups containing approximately even division of sexes for experiments. WT male mice were purchased commercially and were age matched and distributed randomly across groups. Mice 4 weeks of age were used in all experiments. All mouse experiments were performed under animal biosafety level 2 or 3 conditions, as appropriate.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
At 24 hpi the draining popliteal lymph node from mock- or CHIKV-inoculated mice were pooled into individual replicates (3 replicates; LNs from 5 mice pooled per replicate). Lymph nodes were mechanically homogenized using a 22G needle in Click’s medium (Irvine Scientific, 9195) supplemented with 5 mg/mL liberase DL (Roche, 05401160001) and 2.5 mg/mL DNase (Roche 10104159001) for 1 h at 37C. After incubation, digested tissues were clarified by passing through a 100 μm cell strainer. Cell suspensions were enriched for CD45- cells by labeling cells with PE-conjugated anti-mouse CD45 (30-F11), CD140A (APA5), and Ter119 (Ter119) monoclonal antibodies and subsequent depletion of PE-labeled cells using Miltenyi anti-PE microbeads (130-048-801) and Miltenyi MACS LS (130-042-401) columns according to the manufacturer’s instructions with the following modifications: (1) we used 25% of the recommended volume of anti-PE microbeads and (2) we subjected the CD45- enriched cell fraction to a second MACS LS column. All cell suspensions post-column enrichment were enumerated using a hemocytometer. Cell fractions throughout the procedure were analyzed for PE-labeled cell depletion and enrichment of CD45- cells by flow cytometry.
Next GEM single-cell 3′ GEM library and gel bead kit v3.1 (1000128)
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Description |
morrison_count_matrix.tsv.gz
morrison_metadata.tsv.gz
|
| Data processing |
Single-cell RNA-seq libraries were processed using the 10X Genomics cellranger software (5.0.1). Analysis was performed using the R package Seurat (4.0.0). Cell type identification was performed with clustifyr (1.0.0) using references built from published data.
Supplementary_files_format_and_content: Cellranger output files
|
| |
|
| Submission date |
May 18, 2021 |
| Last update date |
Sep 23, 2023 |
| Contact name |
Jay R. Hesselberth |
| E-mail(s) |
jay.hesselberth@cuanschutz.edu
|
| Organization name |
University of Colorado School of Medicine
|
| Department |
Biochemistry and Molecular Gentetics
|
| Lab |
Jay Hesselberth
|
| Street address |
12801 E 17TH AVE
|
| City |
Aurora |
| State/province |
CO |
| ZIP/Postal code |
80045 |
| Country |
USA |
| |
|
| Platform ID |
GPL24247 |
| Series (1) |
| GSE174667 |
MARCO+ lymphatic endothelial cells sequester arthritogenic alphaviruses in the draining lymph node and limit viral dissemination |
|
| Relations |
| Reanalyzed by |
GSM7797750 |
| BioSample |
SAMN19245912 |
| SRA |
SRX10930038 |
