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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Nov 06, 2023 |
| Title |
Mouse replicate 4 Nur77GFP LO [scRNASeq] |
| Sample type |
SRA |
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| Source name |
Lung
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| Organism |
Mus musculus |
| Characteristics |
tissue: Lung
cell line: N/A
cell type: CD4 T cells
genotype: C57BL/6-Tg(Nr4a1-EGFP/cre)820Khog/J
treatment: Mycobacterium tuberculosis infection
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| Growth protocol |
N/A
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
Mycobacterium tuberculosis-infected Nur77-GFP 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 FACS sorted (either GFP-HI or -LO) 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.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic single cell |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
10x Genomics
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| Data processing |
Fastq files from gene expression (GEX) and ADT libraries 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. TCR libraries were processed using Cellranger VDJ
ADT
TotalSeq™-C0157 anti-mouse CD45.2 Antibody
Barcode sequence
CACCGTCATTCAACC
Assembly: mm10
Supplementary files format and content: Tab-separated values files and matrix files
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| Submission date |
Jun 26, 2023 |
| Last update date |
Nov 06, 2023 |
| Contact name |
Tyler Bold |
| E-mail(s) |
tbold@umn.edu
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| Organization name |
University of Minnesota
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| Street address |
2101 6th St SE
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| City |
Minneapolis |
| State/province |
MN |
| ZIP/Postal code |
55455 |
| Country |
USA |
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| Platform ID |
GPL24247 |
| Series (2) |
| GSE235799 |
Activated CD4 T cells in tuberculosis express OX40, a target for host-directed immunotherapy [GFP_HI_LO_scRNASeq] |
| GSE235800 |
Activated CD4 T cells in tuberculosis express OX40, a target for host-directed immunotherapy |
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| Relations |
| BioSample |
SAMN35994688 |
| SRA |
SRX20788151 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7509785_M3_LO_S5_barcodes.tsv.gz |
21.1 Kb |
(ftp)(http) |
TSV |
| GSM7509785_M3_LO_S5_features.tsv.gz |
284.1 Kb |
(ftp)(http) |
TSV |
| GSM7509785_M3_LO_S5_matrix.mtx.gz |
17.8 Mb |
(ftp)(http) |
MTX |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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