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| Status |
Public on Jul 25, 2023 |
| Title |
T cell sci-fate |
| Sample type |
SRA |
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| Source name |
spleen
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| Organism |
Mus musculus |
| Characteristics |
tissue: spleen
cell type: CD8+ T cell
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| Treatment protocol |
Naive CD8 T cells were activated ex vivo with plate-bound anti-CD3 and anti-CD28 in T cell media (TCM) supplemented with 100 U/mL IL-2, 0.5 ng/mL IL-7, 50 ng/mL IL-15, and 0.05 ng/mL IL-12. At days 1, 2, and 4 of activation, two subsequent sci-fate time points were taken as follows: cells were mixed and split into two wells, which had been coated with anti-CD3 and anti-CD28 at day -1 and remained in the incubator with TCM; 4sU was added to one well for a final concentration of 200 µM, and that well was harvested 2 hr later. At that time, 4sU was similarly added to the second well, and that well was harvested 2 hr later. After each 4sU addition, cells were mixed and spun down at 150g for 1 min.
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
Harvested T cells cells were prepared for sci-RNA-seq as described for the sci-fate protocol (Cao et al., Nature Biotech, 2020). Briefly, cells were fixed with ice-cold 4% PFA for 15 min, washed and flash frozen with PBSR [PBS, pH 7.4, 0.2 mg/mL bovine serum albumin (Fisher), 1% SuperRnaseIn (Thermofisher) and 10 mM dithiothreitol (DTT)]. PFA-fixed cells were thawed, washed, and treated with iodoacetamide (IAA) to attach a carboxyamidomethyl group to 4sU.
A single-cell RNA sequencing library was prepared using the sci-RNA-seq protocol (Cao et al., Science, 2017) and NEBNext High-Fidelity 2X PCR Master Mix (NEB) for PCR. Libraries were sequenced on the Illumina NovaSeq system.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic single cell |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Data processing |
All data processing steps were performed as in Cao et al., Nature Biotech, 2020 using the mm10 mouse reference genome for alignment. T > C (sense strand) and A > G (antisense strand) mutations were used to identify reads from newly synthesized transcripts, thus generating two digital gene expression matrices from the full sequencing data and newly synthesized RNA data.
Assembly: mm10
Supplementary files format and content: gene_count.mtx.gz is a sparse matrix file for full transcriptome data: each row corresponds to gene id; each column corresponds to each cell; each value in the matrix corresponds to UMI count.
Supplementary files format and content: df_gene.tsv.gz is the gene annotation file for the full transcriptome data including gene id, gene type and gene short name.
Supplementary files format and content: df_cell.tsv.gz is the cell annotation file for the full transcriptome data: sample is cell id of each single cell with the reverse transcription barcode attached; all_exon are number of UMIs mapping to exon region, all_intron are number of UMIs mapping to intron region, all_reads are number of total UMIs.
Supplementary files format and content: gene_count_newly_synthesized.mtx.gz is a sparse matrix file for newly synthesized transcriptome data: each row corresponds to gene id; each column corresponds to each cell; each value in the matrix corresponds to UMI count.
Supplementary files format and content: df_gene_newly_synthesized.tsv.gz is the gene annotation file for the newly synthesized transcriptome data including gene id, gene type and gene short name.
Supplementary files format and content: df_cell_newly_synthesized.tsv.gz is the cell annotation file for the newly synthesized transcriptome data: sample is cell id of each single cell with the reverse transcription barcode attached; new_exon are number of newly synthesized UMIs mapping to exon region, new_intron are number of newly synthesized UMIs mapping to intron region, newly_syn_UMIs are number of newly synthesized UMIs.
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| Submission date |
Jul 20, 2023 |
| Last update date |
Jul 25, 2023 |
| Contact name |
Kathleen Abadie |
| E-mail(s) |
abadiek@uw.edu
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| Organization name |
University of Washington
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| Department |
Bioengineering
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| Lab |
Kueh Lab
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| Street address |
3720 15th Ave NE
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| City |
Seattle |
| State/province |
Washington |
| ZIP/Postal code |
98195 |
| Country |
USA |
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| Platform ID |
GPL24247 |
| Series (2) |
| GSE237829 |
Flexible and scalable control of T cell memory by a reversible epigenetic switch (sci-fate-seq) |
| GSE237830 |
Flexible and scalable control of T cell memory by a reversible epigenetic switch |
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| Relations |
| BioSample |
SAMN36660394 |
| SRA |
SRX21112753 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7655063_df_cell.tsv.gz |
358.0 Kb |
(ftp)(http) |
TSV |
| GSM7655063_df_cell_newly_synthesized.tsv.gz |
718.1 Kb |
(ftp)(http) |
TSV |
| GSM7655063_df_gene.tsv.gz |
646.4 Kb |
(ftp)(http) |
TSV |
| GSM7655063_df_gene_newly_synthesized.tsv.gz |
574.6 Kb |
(ftp)(http) |
TSV |
| GSM7655063_gene_count.mtx.gz |
141.5 Mb |
(ftp)(http) |
MTX |
| GSM7655063_gene_count_newly_synthesized.mtx.gz |
66.2 Mb |
(ftp)(http) |
MTX |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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