 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Nov 06, 2023 |
| Title |
Mouse replicate 3 |
| Sample type |
SRA |
| |
|
| Source name |
Lung
|
| Organism |
Mus musculus |
| Characteristics |
tissue: Lung
cell line: N/A
cell type: Multiple
genotype: C57BL/6-Tg(Nr4a1-EGFP/cre)820Khog/J
treatment: Mycobacterium tuberculosis infection
|
| Growth protocol |
N/A
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
Mice were euthanized with CO2 and lungs were suspended in a digestion buffer containing DMEM, FBS, Collagenase D, DNase, Heparin, CaCl 2 , and MgCl 2 . Lungs were incubated at 37˚C for 30 minutes and dissociated with a gentleMACS dissociator (Miltenyi Biotec), according to the manufacturer’s protocol. To isolate cells, lung suspensions were filtered through a 70 μm cell strainer. Sorting for the tetramer-enriched scRNA-Seq was performed using a CD4 + T cell isolation kit (Miltenyi Biotec) followed by tetramer staining and enrichment of tetramer-specific cells with anti-PE microbeads (Miltenyi Biotec).
Cells sorted into DMEM with 40% FBS were used for scRNA-Seq sample preparation. After sorting, cell number was confirmed with the Luna II (Logos Biosystems). The appropriate number of cells necessary to achieve a target cell input of 2-10,000 cells was resuspended and used to generate GEMs (Gel Bead-In Emulsion). The Chromium Next GEM Single-cell 5’ Gel beads v2 kit (10X Genomics) was used and manufacturer instructions were followed for GEM generation through cDNA synthesis. A Chromium Control was used for GEM generation and barcoding then a thermocycler was used for cDNA synthesis. cDNA was submitted to the University of Minnesota Genomics Center for sequencing on an Illumina Novaseq. Tetramer-enriched scRNA-seq data pooled from three biological replicates.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic single cell |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Description |
10x Genomics
|
| Data processing |
Fastq files were processed with 10X cellranger using a custom reference genome consisting of the Mus musculis genome mm10 with the addition of the EGFP coding sequence to enable detection of cells expressing GFP reporter gene.
Assembly: mm10
Supplementary files format and content: Tab-separated values files and matrix files
|
| |
|
| Submission date |
Jun 22, 2023 |
| Last update date |
Nov 06, 2023 |
| Contact name |
Tyler Bold |
| E-mail(s) |
tbold@umn.edu
|
| Organization name |
University of Minnesota
|
| Street address |
2101 6th St SE
|
| City |
Minneapolis |
| State/province |
MN |
| ZIP/Postal code |
55455 |
| Country |
USA |
| |
|
| Platform ID |
GPL24247 |
| Series (2) |
| GSE235563 |
Activated CD4 T cells in tuberculosis express OX40, a target for host-directed immunotherapy [Tetramer_scRNASeq] |
| GSE235800 |
Activated CD4 T cells in tuberculosis express OX40, a target for host-directed immunotherapy |
|
| Relations |
| BioSample |
SAMN35839582 |
| SRA |
SRX20748649 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7505353_S4_GFP_barcodes.tsv.gz |
12.8 Kb |
(ftp)(http) |
TSV |
| GSM7505353_S4_GFP_features.tsv.gz |
272.9 Kb |
(ftp)(http) |
TSV |
| GSM7505353_S4_GFP_matrix.mtx.gz |
10.6 Mb |
(ftp)(http) |
MTX |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
|
|
|
|
 |
