 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Jun 13, 2020 |
| Title |
Multiplexed single cell RNA/AbSeq sequencing of ex vivo Treg and ex-Treg from different time points |
| Organism |
Mus musculus |
| Experiment type |
Expression profiling by high throughput sequencing
|
| Summary |
Purpose: identify differentially expressed genes between ex-Treg obtained 5, 10 or 15 days post-adoptive transfer into a lymphopenic environment compared to ex vivo Treg in order to identify Treg subset with heightened instability.
Method: Treg from Foxp3YFP-Cre Rosa26RFP (PMID: 18387831, PMID: 17171761) were co-injected with congenically-disparated naive T cells in a 1:1 ratio into Rag1KO mice and ex-Treg were sorted at 15 days, 10 days or 5 days post-transfer. As a control, ex vivo Treg were sorted from Foxp3YFP-Cre Rosa26RFP (d0 sample). Next, samples were stained with multiplexing antibodies using the BD Single-Cell Multiplexing Kit (BD biosciences), then pooled and stained with the following oligonucleotide-conjugated antibodies: anti‑CD25 (PC61), anti-CD44 (IM7), anti-CD62L (MEL-14), anti-GITR (DTA-1), anti-CD4 (RM4-5), anti‑TCRb (H57-597), anti‑CD69 (H1.2F3), anti‑TIGIT (1G9), anti‑CD95 (Jo2), anti‑CD122 (TM‑1β), anti‑Tim-3 (5D12), anti‑CCR7 (4B12), anti‑CD103 (M290), anti‑CD279 (J43), anti‑CD274 (MIH5), anti‑CD233 (C9B7W), anti‑CD71 (C2), anti‑CD278 (7E7G9), anti‑ITGB7 (M293), anti‑CD137 (1AH2), anti‑CD40 (3/23), anti‑CD3e (145-2C) from BD Biosciences. Single-cell capture and cDNA library preparation was performed using the BD Rhapsody Single-cell analysis system (BD Biosciences), according to the manufacturer’s instructions, using a custom gene panel (591 genes). Sequencing was performed on an Illumina NextSeq500 instrument using a Mid‑Output kit v2.5 (150 cycles, paired-end). Sample demultiplexing, barcode processing, alignment, filtering, UMI counting were done using the standard BD Biosciences Rhapsody analysis pipeline on Seven Bridges (www.sevenbridges.com).
Result: In this study, we showed that ex-Treg downregulate Treg core-specific genes immediatly upon adoptive transfer into a lymphopenic environment. Further, we identified a Treg population enriched for unstable Trg clones.
Conclusion: Our study was able to identify a Treg subset enriched for unstable Treg with plastic potential. This Treg subset appears highly enriched for naïve peripheral-induced Treg.
|
| |
|
| Overall design |
ex vivo Treg and ex-Treg 5, 10 or 15 days post-adoptive transfer into Rag1KO mice were sorted, stained for multiplexing antibodies and stained for Abseq antibodies. Cell capture and single cell RNA sequencing was performed for a custom gene panel using the BD Rhapsody and Illumina NextSeq500 instruments.
|
| |
|
| Contributor(s) |
Junius S, Lemaitre P, Mavrogiannis AV, Liston A, Schlenner SM, Kowalczyk E |
| Citation(s) |
34301799 |
| |
| Submission date |
Jun 12, 2020 |
| Last update date |
Aug 15, 2021 |
| Contact name |
Susan M Schlenner |
| E-mail(s) |
susan.schlenner@kuleuven.be
|
| Organization name |
KU Leuven
|
| Department |
Microbiology, Immunology and Transplantation
|
| Lab |
Adaptive Immunity
|
| Street address |
Herestraat 49
|
| City |
Leuven |
| ZIP/Postal code |
3000 |
| Country |
Belgium |
| |
|
| Platforms (1) |
| GPL19057 |
Illumina NextSeq 500 (Mus musculus) |
|
| Samples (1) |
|
| Relations |
| BioProject |
PRJNA639149 |
| SRA |
SRP267116 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE152387_4_cell_types.txt.gz |
314 b |
(ftp)(http) |
TXT |
| GSE152387_RAW.tar |
7.0 Mb |
(http)(custom) |
TAR (of CSV, FASTA) |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
|
|
|
|
 |
