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| Status |
Public on Jan 11, 2023 |
| Title |
Primary T cells, cytokine-derived cDNA |
| Sample type |
SRA |
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| Source name |
PBMC
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| Organism |
Homo sapiens |
| Characteristics |
tissue: PBMC
cell type: Total T cells
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| Treatment protocol |
PBMC were stimulated with anti-CD3/CD28 Dynabeads for different duration as indicated.
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| Growth protocol |
Cryopreserved PBMC were recovered overnight before experiments
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
For end-timepoint measurement of cytokine secretion by TRAPS-seq, human lymphocytes with depletion of CD19+ cells were enriched using Moflo Astrios cell sorter (Beckman Coulter) and kept in incubator overnight before stimulation. Cells were activated by anti-CD3/CD28 Dynabeads (Gibco, 11161D) at 2:1 bead-to-cell ratio for indicated time periods (0 hour, 1 hours, 6 hours and 16 hours) in 96-well U-bottom plate. Post beads removal, cells were stained with saturated cytokine-capturing antibodies for 15 min at 4oC and resuspended in full medium at 40,000 cells per 10 ml medium in 15 ml Falcon tube. The cytokine capture was carried out at 37oC for 45 min with gentle rotation. Cells that are stimulated for different duration (0, 1, 6, 16 hours) were individually labelled with β2M-targeting hashtags (0.5 µg each Ab-Oligo) and Fc-blocking agents. While indicated, control cells without CapAb modification were labelled with CD45-targeting hashtags instead, which was used to verify potential perturbation of gene expression in cell groups modified with CD45-anchored CapAb. The barcoded cell samples were pooled together and stained with Ab-Oligo cocktail (CD3, CD4, CD8, CD45RA, CCR7, CD27, CD28, CD95, CD69, CD107a, IFN-γ, IL-2 and TNF-α) for 45 min at 4oC. Cells were washed with cold PBS containing 2% FBS for 4 times and re-suspended in PBS before run on the Chromium Controller (10x Genomics). For secretion tracing, total T cells were enriched by immunomagnetic negative selection (STEMCELL Technologies, 19051) and loaded onto the nanowell array dropwise at 40,000 cells per array. The unloaded cells were gently rinsed off and 20 µl anti-CD3/CD28 Dynabeads (Gibco, 11161D) were loaded. The cells were activated in open array for 10 hours before subjected to TRAPS-seq-powered on-array secretion tracing. Briefly, for 1st-round SCA, buffer exchange was performed on the cells within the array before the cells are labelled with unsaturated concentration of CapAbs and Fc-blocking agent for 20 min at 4oC. After buffer exchange, the array was reversibly sealed by glass slide and incubated at 37oC for 45 min. The glass slide was then removed and on-array cell washing was performed. Captured cytokines were stained by the 1st-set of detection antibodies (2 µg per Ab-Oligo against IFN-γ, IL-2 or TNF-α) with further blocking of unbound cytokines with purified unconjugated antibodies. The steps of CapAbs labelling and cytokine capture were repeated one more time. Cells were recovered and stained with pooled 2nd-set of detection antibodies (0.4 µg per cytokine Ab-Oligo against IFN-γ, IL-2 or TNF-α and 0.5 µg per surface marker Ab-Oligo against CD3, CD4, CD8, CD45RA, CCR7, CD27, CD28, CD95, CD69, CD107a). Cells were washed with cold PBS containing 2% FBS for 4 times and re-suspended in PBS before run on the Chromium Controller (10x Genomics).
The 10x Genomics single cell 3' reagent kits (v3.1 Chemistry) was used to generate single-cell barcoded sequencing libraries following reverse transcription. The recovered material includes complementary DNA (cDNA) of mRNA and protein libraries representative of cytokines, surface markers and sample-indicating hashtags. After sample cleanup using silane beads, 35 µl of the eluted DNA sample was pre-amplified following the standard protocol with the addition of two additional primers for surface protein and hashtag and cycled as follows: as follows: 98oC 3 min, 12 cycles of: 98oC 15 s, 63oC 20 s, and 72oC 1 min; Then an extension step of 1 min at 72oC. The 1st-round PCR product was cleaned up and followed by a double size selection step using SPRIselect reagent (Beckman Coulter) to separate protein library from cDNA library. The amplified cDNA product was subjected to final gene expression library construction according to the instructions with the kit. Part of the protein library product (5 µl) was used for the preparation of sequencing libraries corresponding to surface marker and cytokine while additional part (5 µl) was used for the construction of sequencing library of sample hashtags using KAPA HiFi Master Mix (Roche). The 2nd-round P7/read 2 PCR primers used for protein library preparation (the P5/read 1 primer is similar to what was used in cDNA library). The size range and concentration of final library constructions was verified by Agilent 2100 Bioanalyzer. The libraries for cDNA and sample hashtag were pooled and sequenced in Illumina HiSeq platform. The protein libraries for surface protein and cytokine were sequenced in MiSeq platform using MiSeq Reagent Kit v3 and customized sequencing primers.
OTHER: TRAPS-seq
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| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
HiSeq X Ten |
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| Description |
Cytokine-derived oligonucleotide library: read1 file contains cell barcode and UMI; read2 file contains cytokine-targeted antibody derived tag reads
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| Data processing |
Raw mRNA-derived cDNA FASTQ data was processed for cell barcodes calling and UMI counting of mRNA library using Cell Ranger v4.0 (10x Genomics) with default parameters. The generated cell barcodes were used to find individual cell barcode-matched expression profile of sample hashtags, surface proteins and cytokines using CITE-seq-Count with a Hamming distance of 1 for both cell barcodes calling and UMI counting.
Further processing was done with Seurat package (v4.0). Dead cells were removed by filtering out cells expressing > 5% mitochondrial transcript counts.
For the analysis of cytokine secretion, where indicated, sparse dots above 99 quantiles of the background-counts distribution at “0 hr” and apparently discrete dots within data of other time points were removed.
Assembly: hg38
Supplementary files format and content: 10x Genomics output files: barcodes.tsv.gz, features.tsv.gz, matrix.mtx.gz; CITE-seq-Count output files: barcodes.tsv.gz, features.tsv.gz, matrix.mtx.gz.
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| Submission date |
Apr 12, 2022 |
| Last update date |
Jan 12, 2023 |
| Contact name |
Tongjin WU |
| Organization name |
National University of Singapore
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| Department |
Institute for Health Innovation and Technology
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| Street address |
MD6, 14 Medical Drive
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| City |
Singapore |
| ZIP/Postal code |
117599 |
| Country |
Singapore |
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| Platform ID |
GPL20795 |
| Series (1) |
| GSE200690 |
Time-resolved assessment of single-cell protein secretion by sequencing |
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| Relations |
| BioSample |
SAMN27548465 |
| SRA |
SRX14835582 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM6042329_Tracing_ProcessedData_Tracing_Cytokine_barcodes.tsv.gz |
17.0 Kb |
(ftp)(http) |
TSV |
| GSM6042329_Tracing_ProcessedData_Tracing_Cytokine_features.tsv.gz |
140 b |
(ftp)(http) |
TSV |
| GSM6042329_Tracing_ProcessedData_Tracing_Cytokine_matrix.mtx.gz |
74.7 Kb |
(ftp)(http) |
MTX |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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