|
| Status |
Public on Dec 13, 2020 |
| Title |
tSCRB_MM_D8_arm_10 |
| Sample type |
SRA |
| |
|
| Source name |
tSCRB-seq P14s from day 8 acute infection 10
|
| Organism |
Mus musculus |
| Characteristics |
tissue: spleen
cell type: gp33-specific T cell receptor transgenic P14 CD8 T cells
time: Day8
infection: acute LCMV Armstrong infection
sample group: activated P14 T cells
replicate: 10
|
| Extracted molecule |
total RNA |
| Extraction protocol |
the single-cell transcriptomes are generated with a T-cell tailored version of the SCRB-seq protocol using Qiagen TCL buffer supplemented with β-mercaptoethanol for cell lysis and a template switching oligo with locked nucleic acid
[Purification of mouse and human PBMCs] Mouse and human lymphocytes were isolated from a mouse spleen and human blood with the use of Lympholyte M (Cedarlane) and Ficoll-Paque PLUS (GE Healthcare) density gradient media respectively. Cells were washed with media and sorted on BD FACS Fusion (100-micron nozzle standard operation settings single-cell purity) where individual cells meeting the gating strategy were sorted in a tube containing media and used immediately for generating Drop-seq single-cell transcriptomes.
[Purification of naive P14 T cells] Single cell splenocyte suspensions were obtained by mashing total spleens through a 100 μm nylon cell strainer (BD Falcon) and lysing red blood cells with a hypotonic ACK buffer. Naïve transgenic P14 CD8 T-cells were isolated using the mouse CD8+ T-cell enrichment kit (Miltenyi Biotech Bergisch-Gladbach Germany). Surface staining was performed for 40 min at 4°C in supplemented DMEM media (Gibco ThermoFisher Scientific) using the following antibodies: anti-CD8a (53-6.7 eBioscience) CD4 (RM4-4 Biolegend) and Vα2 (V20.1 eBioscience). Cells were washed twice with media and sorted on BD FACS Fusion (100-micron nozzle standard operation settings single-cell purity). Individual cells meeting the gating strategy were sorted in tube containing media (Drop-seq) and used immediately or directly in lysis buffer into individual wells of a low-binding PCR plate (SCRB-seq) which was subsequently spun down snap-frozen on dry ice and stored at -80oC until use. All data were analyzed using FlowJo (TreeStar).
[Purification of activated P14 T cells] Single cell splenocyte suspensions were obtained by mashing total spleens through a 100 μm nylon cell strainer (BD Falcon) and red blood cells were lysed with a hypotonic ACK buffer. Naïve transgenic P14 CD8 T-cells were isolated using the mouse CD8+ T-cell enrichment kit (Miltenyi Biotech Bergisch-Gladbach Germany). 2x103 CD45.1+ P14 TCRαβ were transferred into naïve CD45.2+ C57BL/6 mice followed by their infection. On the day of the sort single cell splenocyte suspensions were obtained as described above. Activated transgenic P14 CD8 T-cells were isolated using anti-CD45.1 biotin/anti-biotin conjugated microbeads and magnetic MACS cell separation (Miltenyi Biotech Bergisch-Gladbach Germany). Surface staining was performed for 40 min at 4°C in supplemented DMEM media (Gibco ThermoFisher Scientific) using the following antibodies: anti-CD8a (53-6.7 eBbioscience) CD4 (RM4-4 Biolegend) CD45.1 (A20 eBioscience) and CD45.2 (104 eBioscience). Cells were washed twice with media and sorted on BD FACS Fusion (100-micron nozzle standard operation settings single-cell purity). Individual cells meeting the gating strategy were sorted in tube containing media (Drop-seq) and used immediately or directly in lysis buffer into individual wells of a low-binding PCR plate (SCRB-seq) which was subsequently spun down snap-frozen on dry ice and stored at -80oC until use. All data were analyzed using FlowJo (TreeStar).
[Drop-seq library construction protocol] The original Drop-seq protocol was performed as described by Macosko and colleagues16. All runs were performed at 3000 µl/hour cell and bead rates with the use of Drop-seq PDMS device (Nanoshift). The following reagents were used for the Drop-seq modification described in this article: Lithium dodecyl sulfate (L9781 Sigma-Aldrich) Lithium chloride (62476 SigmalAldrich) Igepal (I8896-50ML Sigma-Aldrich) NaCl (S3014 Sigma-Aldrich) 3âLNA(G)TSO (IDT) Maxima H Minus RT (EP0753 ThermoFisher) SuperScript IV RT (18090010 ThermoFisher) SMARTScribe RT (639536 Takara Bio) and Ficoll PM 400 (GE17-0300-10 GE Healthcare). After RNA purification and reverse transcription the generated single-cell cDNA was amplified for 18 cycles. The barcoded single-cell amplicons were purified with the use of (0.6x) Agencourt AMPure XP beads. 1 ng of the resulting amplified cDNA were used for library preparation with the Illumina Nextera XT DNA Library reagents (FC-131-1024 Illumina). After PCR amplification of the fragmented libraries the samples were purified with (0.6x) Agencourt AMPure XP beads and eluted in 10 µl of molecular grade water. The quality of the resulting library was assessed with the use of Agilent High Sensitivity DNA Kit (5067-4626 Agilent). The library quantification was performed based on the Illumina recommendations (SY-930-1010 Illumina) with the use of KAPA SYBR FAST qPCR Master Mix (KK4600 Kapa Biosystems). The samples were sequenced on Illumina HiSeq 2500 system at the following conditions - rapid run paired-end 20 bp read 1 45 bp read 2 single-indexed sequencing resulting in 0.5 million reads per single-cell.
[SCRB-seq library construction protocol] The original SCRB-seq protocol was performed as described by Soumillon and colleagues17. To allow profiling of T cells we introduced a step of RNA purification before reverse transcription with the use of RNAClean XP (A63987 Beckman Coulter). This was used for all modifications assessed in this paper. The following reagents were used for the SCRB-seq modification described in this article: Triton X-100 (T9284-100ML Sigma-Aldrich) Buffer TCL (1031576 Qiagen) 2-Mercaptoethanol (M3148-25ML Sigma-Aldrich) 3âLNA(G)TSO (IDT) and Betaine solution (B0300-1VL Sigma-Aldrich). After reverse transcription the single-cell transcriptomes were amplified without pooling for 20 cycles. The barcoded single-cell amplicons were purified with the use of (0.6x) Agencourt AMPure XP beads and libraries were generated as for Drop-seq. The samples were sequenced on Illumina HiSeq 2500 system at the following conditions - rapid run paired-end 16 bp read 1 49 bp read 2 single-indexed sequencing resulting in 0.5 million reads per single-cell.
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| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Data processing |
dropSeqPipe 0.4. The pipeline includes filtering mapping and cell/UMI demultiplexing. Each protocols has it's own supplementary configuration file describing all the paramters used.
Filtering is done by cutadapt
Mapping is done by STAR
Demultiplexing is done by drop-seq tools v2
Supplementary_files_format_and_content: custom_adapter.fa - Adapters used in the protocols. Used to trim data in the pipeline
gtf_biotypes.yaml - List of biotypes used for mapping with STAR.
DS_config.yaml - Drop-seq configuration file with all the parameters used in the pipeline
SCRB_config.yaml - scrb-seq configuration file with all the parameters used in the pipeline
SCRB_barcodes csv - List of cell barcodes in the SCRB-seq protocols used in the pipeline
barcodes.tsv.gz - Cell barcodes with prefix
features.tsv.gz - Genes
matrix.mtx.gz - MTX matrix of UMI counts
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| |
|
| Submission date |
Dec 12, 2020 |
| Last update date |
Dec 18, 2020 |
| Contact name |
Patrick Roelli |
| E-mail(s) |
patrick.roelli@gmail.com
|
| Organization name |
TUM
|
| Department |
Tierphysiologie & Immunologie
|
| Lab |
Dr. Zehn
|
| Street address |
Weihenstephaner Berg 3
|
| City |
Freising |
| ZIP/Postal code |
85354 |
| Country |
Germany |
| |
|
| Platform ID |
GPL17021 |
| Series (1) |
| GSE163089 |
Tailoring the resolution of single-cell RNA sequencing for primary cytotoxic T cells |
|
| Relations |
| BioSample |
SAMN17068644 |
| SRA |
SRX9678717 |
