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| Status |
Public on Aug 10, 2023 |
| Title |
CD4 memory T cells, cow milk allergic and non-allergic pediatric subjects, Gene expression, tube5B |
| Sample type |
SRA |
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| Source name |
Blood
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| Organism |
Homo sapiens |
| Characteristics |
tissue: Blood
cell type: CD4 memory T cells
treatment: Cow milk epitope pool
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| Treatment protocol |
PBMC stimulated with cow milk epitope pool (M111) for 6 hours and then sorted for CD4 memory T cells either positive for CD154, CD137, both (Positive) or none (Negative)
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| Extracted molecule |
total RNA |
| Extraction protocol |
For single cell RNA-seq assays, we used the 10x Genomics platform. For each patient sample, the maximum amount of CM+ memory T cells and 15,000 CM- cells were collected in low retention and sterile ice-cold 1.5 mL tubes containing 500 μL of PBS - FBS (1:1 volume) completed with RNase inhibitor (1:100). Each experiment of 12 patients generated 4 tubes where each combined the CM- fraction of 3 subjects with the CM+ fraction of 3 different subjects. Approximately 50,000 sorted cells per tube were processed and loaded on the 10X Chromium Controller (10X Genomics). We used a 5’mRNA capture chemistry (5’ 10X v2 chemistry) and performed cDNA amplification and library preparation for gene expression, feature barcoded surface antibodies, and TCR according to manufacturer's protocol.
Final libraries were quality checked for fragment size by capillary electrophoresis (Fragment analyzer), and quantity by fluorescence assay (Picogreen) before pooling and sequencing on a NovaSeq 6000 (200 cycle S4 kit v1.5; Illumina, San Diego, CA, USA) to a depth of > 25,000 reads/cell for GEX, > 5,000 reads/cell for hashtag oligo libraries, and >5,000 reads/cell for TCR libraries.
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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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| Description |
polyA RNA
10x Genomics
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| Data processing |
Data processing was done using Cell Ranger software v5.0.0.
The illumina barcode demultiplexing was performed using mkfastq
Alignment with reference GRCh38 (GENCODE v32/Ensembl 98) and gene counting were done using Multi program
R package Seurat was used for downstream clustering and UMAP analysis
Seurat's function MultiSeqDemux was used for hashtag demultiplexing and singlet assignment
Assembly: GRCh38 (GENCODE v32/Ensembl 98)
Supplementary files format and content: 10x Genomics output files: barcodes.tsv.gz, features.tsv.gz, matrix.mtx.gz
Supplementary files format and content: seurat metadata
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| Submission date |
May 03, 2023 |
| Last update date |
Aug 10, 2023 |
| Contact name |
Ashu Sethi |
| E-mail(s) |
ashu@lji.org
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| Organization name |
La Jolla Institute for Immunology
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| Department |
Health Sciences
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| Street address |
9420 Athena Circle
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| City |
San Diego |
| State/province |
CA |
| ZIP/Postal code |
92037 |
| Country |
USA |
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| Platform ID |
GPL24676 |
| Series (1) |
| GSE231590 |
Identification of cow milk epitopes to characterize and quantify disease-specific T cells in allergic children |
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| Supplementary file |
Size |
Download |
File type/resource |
| GSM7291308_FeSo05_Hu_6.barcodes.tsv.gz |
54.6 Kb |
(ftp)(http) |
TSV |
| GSM7291308_FeSo05_Hu_6.features.tsv.gz |
325.8 Kb |
(ftp)(http) |
TSV |
| GSM7291308_FeSo05_Hu_6.matrix.mtx.gz |
94.4 Mb |
(ftp)(http) |
MTX |
| Raw data not provided for this record |
| Processed data provided as supplementary file |
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