|
| Status |
Public on Jan 01, 2024 |
| Title |
scTCR-seq_Donor allo |
| Sample type |
SRA |
| |
|
| Source name |
Donor allo
|
| Organism |
Mus musculus |
| Characteristics |
phenotype: transgenic FoxP3EGFP mice (B6; CD45.2; H-2b)
Sex: female
cell type: Donor Treg allo reisolated from Donor allo
cell markers: Treg-CD4+CD25highCD62L+
|
| Treatment protocol |
Prophylactic treatment of GvHD: Single cell suspensions from BM (femora and tibiae of both hind legs) and spleen were prepared. T cells were depleted from BM (T cell depleted bone marrow; TCDBM) by using anti-CD90.2 MicroBeads with the MidiMACS® system (Miltenyi Biotec). CD4+CD25- conventional T cells (Tconv) were isolated from splenocytes by depletion of CD25+ cells using anti-CD25-PE and anti-PE UltraPure MicroBeads (Miltenyi Biotec), followed by enrichment of CD4+ cells from the CD25- fraction through labeling with anti-CD4 MicroBeads (Miltenyi Biotec) using the MidiMACS® system. For short-term (d7) FACS and RNA-seq experiments BALB/c recipients were lethally irradiated (8 Gy) on the day of BMT and transplanted i.v. with 2.5 x106 TCDBM cells and 1x106 Tconv from B6-CD45.1 mice with or without in vitro expanded alloTreg from Foxp3gfp mice at a 1:1 ratio.
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| Growth protocol |
Donor Treg Expansion: Splenocytes from transgenic FoxP3EGFP mice (B6; CD45.2; H-2b) were stained with anti-CD25-PE and anti-PE UltraPure MicroBeads and enriched for CD25+ cells by MACS® (Miltenyi Biotec). The CD25+ fraction was further stained for CD4, CD8 and CD62L and Treg were sorted on a FACSAriaTM IIu or FACSAriaTM Fusion (Becton Dickinson) as CD4+CD25highCD62L+ cells; purity of sorted cells was >98%. For allospecific in vitro expansion, sorted Treg were seeded at 5x104/well in cDMEM together with 2,5x104/well (d0) or 1x104/well (restimulation on d7) anti-CD11c MicroBead-enriched (Miltenyi Biotec) and irradiated (30 Gy) CD11c+ DC from BALB/c mice. Cells received fresh medium on d4 and d10 and were harvested on d12.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
For re-isolation of donor Treg on day 7 after transplantation, single cell suspensions from spleen, liver and colon were prepared, stained for H-2Kb, CD45.1, CD45.2, TCRβ and CD4 and sorted on a FACSAria IIuTM or a FACSAriaTM Fusion.
ScRNA-seq libraries were constructed using 13-16 cycles of PCR. Products were purified using Ampure XP beads and quality was controlled using Agilent Tapestation. ScTCR-seq libraries were generated from the same cDNAs using the Single Cell V(D)J Enrichment Kit, Mouse T Cell (10x Genomics).
ScRNA-libraries were sequenced using single-read sequencing (S1 flow cell, 100 bp) on the Illumina NovaSeq™. scTCR-libraries were sequenced on the Illumina NextSeq 550 with 150 cycles single-read sequencing.
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| |
|
| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
NextSeq 550 |
| |
|
| Data processing |
ScRNA:Sequencing reads were demultiplexed using cellranger mkfastq (version 4.0.0),scTCR:Fastq files were processed using cellranger (version 5.0.0)
ScRNA:The reads were subsequently mapped to the mouse genome using cellranger count (version 4.0.0).
ScRNA: Quality control and analysis was performed with the Seurat package (v4.0.0) in R.
scTCR:Fastq files were processed using cellranger (version 5.0.0)
ScTCR:The reads were subsequently mapped to the mouse genome using cellranger count (version 5.0.0).
ScTCR: Quality control and analysis was performed with the Seurat package (v5.0.0) in R
Assembly: ScRNA: Mouse reference, mm10 (GENCODE vM23/Ensembl98),scTCR: mm10 reference genome (refdata-cellranger-vdj-GRCm38-alts-ensembl-5.0.0
Supplementary files format and content: cellranger output, rds (R.4.1.1), loom (python 3.8.13), H5ad (python 3.8.13)
Library strategy: scTCR-seq
|
| |
|
| Submission date |
Jan 26, 2023 |
| Last update date |
Jan 01, 2024 |
| Contact name |
Michael Rehli |
| E-mail(s) |
michael.rehli@klinik.uni-r.de
|
| Organization name |
University Hospital Regensburg
|
| Department |
Internal Med III
|
| Street address |
F.-J.-Strauss-Allee 11
|
| City |
Regensburg |
| ZIP/Postal code |
93042 |
| Country |
Germany |
| |
|
| Platform ID |
GPL21626 |
| Series (2) |
| GSE223798 |
Donor regulatory T cells rapidly adapt to recipient tissues to control acute graft-versus-host disease [scRNA-seq & scTCR-seq] |
| GSE223800 |
Donor regulatory T cells rapidly adapt to recipient tissues to control acute graft-versus-host disease |
|
| Relations |
| BioSample |
SAMN32932798 |
| SRA |
SRX19192134 |
