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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Jun 13, 2020 |
| Title |
D0 Treg, D5/10/15 ex-Treg |
| Sample type |
SRA |
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| Source name |
splenocytes + lymph nodes pooled
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| Organism |
Mus musculus |
| Characteristics |
strain name: Foxp3-YFP-Cre Rosa26 flSTOPfl RFP
gender: male
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| Treatment protocol |
Treg of Foxp3 YFP-Cre Rosa26 fl-STOP-fl RFP mice and naïve T cells of congenically disparated mice were sorted and adoptively transferred into Rag1-KO mice.
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| Extracted molecule |
total RNA |
| Extraction protocol |
At day 5, day 10 and day 15 recipient mice were euthanized and spleen and lymp nodes were processed and FACS sorted for ex-Treg. As a control ex vivo Treg on day 0 were sorted from Foxp3YFP-Cre Rosa26 fl STOP fl RFP mice. Next, samples were stained using the with sample tag antibodies (BD Single-Cell Multiplexing Kit, BD biosciences). Afterwards, samples were pooled and stained using the following oligonucleotide-conjugated antibodies according to manufacturing instructions: anti CD25 (PC61), anti-CD44 (IM7), anti-CD62L (MEL-14), anti-GITR (DTA-1), anti-CD4 (RM4-5), anti TCR (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 CD3 (145-2C) from BD Biosciences. Single-cell capture and cDNA library preparation was performed using the BD Rhapsody Single-cell analysis system (BD Biosciences)
Library was constructed accordingly to BD's instruction (Doc ID: 214508 Rev 3.0). Briefly, captured mRNA were amplified with with a custom primer panel and with specific primer for the BD AbSeq and Sample tag antibodies. The Sample Tag and BD AbSeq PCR products were separated from the mRNA targeted PCR products by double-sided size selection with Agencourt® AMPure® XP magnetic beads. The BD AbSeq and Sample Tag PCR products underwent a separate index PCR from mRNA products with library index primers. BD Rhapsody™ mRNA targeted PCR products and Sample Tag PCR products underwent a second PCR amplification followed by an index PCR with library index primers.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
multiplexed sample
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| Data processing |
The raw data were processed thought BD Rhapsody pipeline. All steps in details are documented here: https://scomix.bd.com/hc/article_attachments/360048823252/23-21713-01_Rhapsody_Bioinfo_Handbook-ruo.pdf,
Briefly, the raw data were filtered from low quality reads based on average Phred score <20. R1 reads were used to determine the cell entity and molecule count. R2 reads were aligned to BD custom reference to define mRNA origin. Information about cell label, molecule and mRNA target was combined from R1 and R2. The molecule count was corrected by applying RSEC and DBEC correction for all genes that reached sequencing saturation >4. The residual cell labels were filtered based on cumulative number of mRNA reads in cells.
Genome_build: BD custom reference file
Supplementary_files_format_and_content: csv files generated by BD Rhapsody pipeline, expression matrix with molecule counts per gene/per cell index, sample tag call file with anotated name of sample per cell index
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| Submission date |
Jun 12, 2020 |
| Last update date |
Jun 13, 2020 |
| Contact name |
Susan M Schlenner |
| E-mail(s) |
susan.schlenner@kuleuven.be
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| Organization name |
KU Leuven
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| Department |
Microbiology, Immunology and Transplantation
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| Lab |
Adaptive Immunity
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| Street address |
Herestraat 49
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| City |
Leuven |
| ZIP/Postal code |
3000 |
| Country |
Belgium |
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| Platform ID |
GPL19057 |
| Series (1) |
| GSE152387 |
Multiplexed single cell RNA/AbSeq sequencing of ex vivo Treg and ex-Treg from different time points |
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| Relations |
| BioSample |
SAMN15224445 |
| SRA |
SRX8538481 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4614203_Pool3319_DBEC_MolsPerCell.csv.gz |
609.8 Kb |
(ftp)(http) |
CSV |
| GSM4614203_Pool3319_Sample_Tag_Calls.csv.gz |
24.5 Kb |
(ftp)(http) |
CSV |
| GSM4614203_antibodies_ref_seq.fasta.gz |
711 b |
(ftp)(http) |
FASTA |
| GSM4614203_cell_labels_ref_seq.fasta.gz |
6.2 Mb |
(ftp)(http) |
FASTA |
| GSM4614203_sample_tags_ref_seq.fasta.gz |
201 b |
(ftp)(http) |
FASTA |
| GSM4614203_targeted_panel_ref_seq.fasta.gz |
74.9 Kb |
(ftp)(http) |
FASTA |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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