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| Status |
Public on Aug 06, 2019 |
| Title |
IL-4+ gene expression library |
| Sample type |
SRA |
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| Source name |
Sorted IL-4 4GET+ Activated CD4 T cells
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| Organism |
Mus musculus |
| Characteristics |
strain/background: C57BL/6 4GET IL-4 Reporter
treatment: Immunization with lternaria alternata + NP-OVA
tissue: Mediastinal Lymph Node
cell type: IL-4+ activated CD4 T cells
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| Treatment protocol |
Mice were immunized with Alternaria alternata extract (10μg) + NP-OVA (25μg) intranasally and day 7 mediastinal lymph node Tfh cells or IL-4+ activated CD4 T cells were sorted and stimulated with PMA and Ionomycin for 30 min and then subjected to single-cell RNA sequencing using the 10X platform and the reads were analyzed.
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| Growth protocol |
NA
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| Extracted molecule |
total RNA |
| Extraction protocol |
Sorted cells from each mouse sample were labeled with cell hashing antibodies (BioLegend TotalSeq anti-mouse Hashtag #3-6). 12,000 cells (4000 cells from each hashtagged mouse) were loaded into one lane of a 10X Chromium microfluidic chip. Single cell capture, barcoding and library preparation were performed using the 10X Chromium platform, version 2 chemistry for the Tfh sort and version 3 chemistry for the IL-4 4Get+ sort. cDNA and libraries were checked for quality on Agilent 4200 Tapestation, quantified by KAPA qPCR, and pooled using a ratio of 95% gene expression library and 5% hashtag library before sequencing, one on a single lane of an Illumina HiSeq4000 and the other on 16.67% of lane of an Illumina NovaSeq 6000 S2 flow cell, both targeting 6,000 barcoded cells with an average sequencing depth of 50,000 reads per cell.
Chromium System (10X Genomics) v2 or v3 chemistry.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
AW19003
PolyA RNA
Single-cell RNA-seq
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| Data processing |
FASTQ files corresponding to AW18001 and AW18002 were generated and demultiplexed using Illumina bcl2fastq 2.18.0.12. Similarly, FASTQ files for AW19003 and AW19004 were generated and demultiplexed using Illumina bcl2fastq 2.20.0.422.
For samples AW18001 and AW18002, R1 contains a 16bp cell barcode + 10bp UMI barcode. For samples AW19003 and AW19004, R1 contains a 16bp cell barcode + 12bp UMI barcode. For all samples, R2 contains the biological reads.
The barcodes in AW18001/AW18002 correspond to the same cells, similarly for AW19003/AW19004. Note that gene expression barcodes (AW18001 and AW19003) have a "-1" appended to each barcode that is not present in the hashtag barcodes (AW18002 and AW19003).
A filtered digital gene expression matrix was generated for each gene expression library against the 10X Genomics mm10 reference build (version 2.1.0, GRCm38.84) using 10X Genomics CellRanger count version 2.2.0 for AW18001 and version 3.0.2 for AW19003.
A digital counts matrix was generated for each hashtag library using CITE-Seq-Count (https://github.com/Hoohm/CITE-seq-Count), version 1.3.3 for AW18002 and version 1.4.1 for AW19004. This produces a matrix of counts with hashtag labels for rows (available via BioLegend https://www.biolegend.com/totalseq) and cell barcodes for columns.
Genome_build: GRCm38.84
Supplementary_files_format_and_content: Transcriptomic and HashTag (HTO) counts matrices in Matrix Market CSR format. Rows correspond to genes in the order provided in genes.tsv/features.tsv and columns correspond to cell-barcodes in the order provided in barcodes.tsv.
Supplementary_files_format_and_content: AW18001_barcodes.tsv: Column labels (cell barcodes) of gene expression counts matrix.
Supplementary_files_format_and_content: AW18001_genes.tsv: Row labels (genes) of gene expression counts matrix.
Supplementary_files_format_and_content: AW18001_matrix.mtx: Digital counts matrix for gene expression (matrix market format).
Supplementary_files_format_and_content: AW18002_hto_counts.csv: Digital counts matrix for HTOs (including row and column labels).
Supplementary_files_format_and_content: AW19003_barcodes.tsv.gz: Column labels (cell barcodes) of gene expression counts matrix.
Supplementary_files_format_and_content: AW19003_features.tsv.gz: Row labels (genes) of gene expression counts matrix.
Supplementary_files_format_and_content: AW19003_matrix.mtx.gz: Digital counts matrix for gene expression (matrix market format).
Supplementary_files_format_and_content: AW19004_hto_barcodes.tsv.gz: Column labels (cell barcodes) of HTO counts matrix.
Supplementary_files_format_and_content: AW19004_hto_features.tsv.gz: Row labels (genes) of HTO counts matrix.
Supplementary_files_format_and_content: AW19004_hto_matrix.mtx.gz: Digital counts matrix for HTOs (matrix market format).
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| Submission date |
Jun 16, 2019 |
| Last update date |
Aug 06, 2019 |
| Contact name |
William F Flynn |
| Organization name |
The Jackson Laboratory
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| Street address |
10 Discovery Drive
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| City |
Farmington |
| State/province |
CT |
| ZIP/Postal code |
06032 |
| Country |
USA |
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| Platform ID |
GPL24247 |
| Series (1) |
| GSE132798 |
Identification of a T follicular helper subset that drives anaphylactic IgE |
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| Relations |
| BioSample |
SAMN12070432 |
| SRA |
SRX6075340 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3892346_AW19003_barcodes.tsv.gz |
38.5 Kb |
(ftp)(http) |
TSV |
| GSM3892346_AW19003_features.tsv.gz |
250.8 Kb |
(ftp)(http) |
TSV |
| GSM3892346_AW19003_matrix.mtx.gz |
45.8 Mb |
(ftp)(http) |
MTX |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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