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| Status |
Public on Oct 17, 2023 |
| Title |
WT (GEX) |
| Sample type |
SRA |
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| Source name |
Spleen
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| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6J background
genotypes: WT
sorting strategy: live CD90+ CD8+ CD44+ T cells
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| Extracted molecule |
total RNA |
| Extraction protocol |
Wildtype and Il1rl1-ExAB-/- mice (n = 3 per genotype) were infected intravenously with LCMV-WE (2x106 PFU). At day 7 post infection, splenocytes were isolated by mechanical disruption and erythrocytes were lysed by incubation in erythrocyte lysis buffer (10 mM KHCO3, 155 mM NH4Cl, 0.1 mM EDTA, pH 7.5). Obtained cell suspensions were passed through 70µm filters and T cells were enriched by depleting CD19+ B cells using magnetic activated cell sorting (MACS, Miltenyi Biotec). Subsequently, live CD90+ CD8+ CD44+ T cells were flowcytometrically sorted using a BD Aria II device, stained with Total-Seq antibodies (Biolegend) and LCMV dCODE dextramers (Immudex) according to manufacturer’s instructions and subjected to the 10x Genomics scRNA-Seq workflow.
See sorting strategy.
10x Genomics workflow for cell capturing. For the preparation of scRNA gene expression (GEX), TCR and CiteSeq libraries the Chromium Next GEM Single Cell 5’ Library & Gel Bead Kit v1.1 as well as the Chromium Single Cell 5' Feature Barcode Library Kit (10x Genomics) were used for CD127 (GTGTGAGGCACTCTT), antiAPC (TTAACCGTCTCCCTT), CD279 (GAAAGTCAAAGCACT), CD197 (TTATTAACAGCCCAC), CD39 (GCGTATTTAACCCGT), CD185 (ACGTAGTCACCTAGT), CD31 (GCTGTAGTATCATGT), KLRG1 (GTAGTAGGCTAGACC), CD183 (GTTCACGCCGTGTTA), CD49d (CGCTTGGACGCTTAA), CD73 (ACACTTAACGTCTGG), FQPQNGQFI (GTGGTCGACCGTCTG), KAVYNFATC (CCTTAATATGTAGAA), SGVENPGGYCL (AGGAACCGCTGTTGA), CSANNSHHYI (TCATGTCAGACTTGG), ISHNFCNL (CGTGCAAGGCCGGAA) as well as , Hashtag1 (ACCCACCAGTAAGAC), Hashtag2 (GGTCGAGAGCATTCA), Hashtag3 (CTTGCCGCATGTCAT), Hashtag4 (AAAGCATTCTTCACG),Hashtag5 (CTTTGTCTTTGTGAG). After cDNA amplification the feature barcode libraries were prepared separately using the Single Index Kit N Set A (10x Genomics). TCR target enrichment was performed using the Chromium Single Cell V(D)J Enrichment Kit for murine T cells (10x Genomics). Final GEX and TCR libraries were obtained after fragmentation, adapter ligation and final Index PCR using the Single Index Kit T Set A (10x Genomics). The Qubit dsDNA HS assay kit (life technologies) was used for library quantification and fragment sizes were determined using the Fragment Analyzer with the NGS Fragment Kit (1-6000bp) (Agilent)
Chromium Next GEM Single Cell 5’ Library & Gel Bead Kit v1.1 as well as the Chromium Single Cell 5' Feature Barcode Library Kit (10x Genomics) were used
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| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
NextSeq 2000 |
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| Description |
total RNA
3 mice, hashtagged
WT_GEX
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| Data processing |
Raw data were processed using cellranger-3.1.0 with refdata-gex-mm10-2020-A and refdata-cellranger-vdj_GRCm38_alts_ensembl-mouse-2.2.0 as reference. .Mkfastq, count and vdj were used in default parameter settings with 3000 expected cells for demultiplexing, detection of intact cells, quantification of gene expression, antibody capture as well as assembly and quantification of T cell receptor sequences.
Assembly: mm10
Supplementary files format and content: h5: aggregated gene count matrix in hdf5 format including antibody counts for CD127, AntiAPC, CD279, CD197, CD39, CD185, CD31, KLRG1, CD183, CD49d, CD73, FQPQNGQFI, KAVYNFATC, SGVENPGGYCL, CSANNSHHYI, ISHNFCNL as well as hashtag counts for different biological replicates. feature_reference.csv: ADT/HTO reference file, fasta: high-confidence contig sequences of immune receptors with the corresponding cellular barcode and contig-id; csv: results from single cell immune profiling, comprising the annotation of the immune receptor, cellular barcode and contig-id.
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| Submission date |
May 24, 2022 |
| Last update date |
Oct 17, 2023 |
| Contact name |
Pawel Durek |
| E-mail(s) |
pawel.durek@drfz.de
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| Organization name |
Deutsches Rheuma-Forschungszentrum
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| Street address |
Charitéplatz 1
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| City |
Berlin |
| ZIP/Postal code |
10117 |
| Country |
Germany |
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| Platform ID |
GPL30172 |
| Series (2) |
| GSE204693 |
scGEX/scTCR-Seq of CD44+ cytotoxic T cells in LCMV-infected Wildtype and Il1rl1-ExAB-/- mice |
| GSE204695 |
A type 1 immunity-restricted promoter of the IL-33 receptor gene drives antiviral T cell responses |
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| Relations |
| BioSample |
SAMN28634993 |
| SRA |
SRX15437177 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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