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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Oct 15, 2019 |
| Title |
CD3+ cells, Naïve (3' V(D)J scRNASeq) |
| Sample type |
SRA |
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| Source name |
CD3+ cells
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| Organism |
Homo sapiens |
| Characteristics |
cell type: CD3+ cells
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| Treatment protocol |
Bulk CD7+ progenitor T-cells (proT-cells) sorted from day 13-14 naïve- or SR1-CD34+/OP9-DL1 cultures were intrahepatically injected into non-irradiated 2-5 day old immunodeficient neonatal mice. Splenocytes were harvested from mice 10–12 weeks after intrahepatic injection proT-cells.
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| Growth protocol |
All mice were maintained in specific pathogen free conditions.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Splenic CD3+ cells were FACS sorted.
10x sample preparation - Samples were prepared as outlined by 10x genomics Single Cell V(D)J Reagent Kits v1 user guide. 31.7ul of the cell suspension was loaded onto the 10x single cell A chip targeting 2400 cells per sample. After droplet generation, samples were transferred onto a pre-chilled 96 well plate (Eppendorf), heat sealed and incubated overnight in a Veriti 96-well thermos cycler (Thermo Fisher) for the reverse transcriptase (RT) and template switching (TS) reactions. Following the RT and TS reaction, cDNA was recovered using the Recovery Agent provided by 10x and subsequently cleaned up using a Silane DynaBead (10x Genomics) mix as outlined by the user guide. Purified cDNA was amplified,cleaned up using SPRIselect beads (Beckman). Samples were run undiluted on a Bioanalyzer (Agilent Technologies) to determine cDNA concentration. For TCR libraries, 2ul of cDNA library was diluted in 33ul of nuclease-free water before undergoing a series of target enrichment reactions. These enriched libraries were then fragmented, ligated and indexed as described in the user guide. For the gene expression library, samples were prepared as outlined by the Single Cell V(D)JReagent Kits v1 user guide with modifications to the PCR cycles based on the calculated cDNA concentration.
Sequencing - The molarity of each library was calculated based on library size as measured bioanalyzer (Agilent Technologies) and qPCR amplification data (Roche). Samples were pooled and normalized to 10 nM, then diluted to 2 nM using elution buffer (Qiagen) with 0.1% Tween20 (Sigma). Each 2 nM pool was denatured using 0.2N NaOH at equal volumes for 5 minutes at room temperature. Library pools were further diluted to 20 pM using HT-1 (Illumina) before being diluted to a final loading concentration of 1.8 pM. 1300ul from the 1.8 pM pool was loaded into Nextseq Midoutput cartridge (Illumina) for cluster generation. 5' expression libraries were sequenced on a Nextseq with the following run parameters: Read 1 - 26 cycles, read 2 - 98 cycles, index 1 - 8 cycles. The enriched V(D)J libraries were sequence paired-end 150 cycles, index 1-8 to a targeted depth of 5,000 read pairs/target cell while the gene expression libraries were sequenced to a targeted depth of 40,000 read pairs/cell.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
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| Data processing |
I1 read: contains the sample index.
R1 read: contains the cell barcode (first 16 nt) and UMI (next 10 nt).
R2 read: contains the RNA sequence.
Filtering (Immunarch using R software)
Genome_build: hg19
Supplementary_files_format_and_content: CSV file containing individual cells (marked by unique barcodes), and their expression of V, D, J and CDR3 genes.
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| Submission date |
Aug 17, 2019 |
| Last update date |
Oct 16, 2019 |
| Contact name |
Jastaranpreet Singh |
| E-mail(s) |
jastaran.singh@utoronto.ca
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| Organization name |
University of Toronto
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| Street address |
2075 Bayview Avenue
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| City |
Toronto |
| State/province |
Ontario |
| ZIP/Postal code |
M4N 3M5 |
| Country |
Canada |
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| Platform ID |
GPL18573 |
| Series (2) |
| GSE135928 |
3' V(D)J scRNASeq of splenic CD3+ cells from immunodeficient mice injected with progenitor T-cells generated from naïve, non-expanded or SR1-expanded human cord blood-derived CD34+ cells |
| GSE135929 |
scRNASeq of splenic CD3+ cells from immunodeficient mice injected with progenitor T-cells generated from naïve, non-expanded or SR1-expanded human cord blood-derived CD34+ cells |
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| Relations |
| BioSample |
SAMN12594277 |
| SRA |
SRX6731992 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4038045_Naive.csv.gz |
106.7 Kb |
(ftp)(http) |
CSV |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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