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| Status |
Public on Jul 31, 2023 |
| Title |
Pt-19_GEX |
| Sample type |
SRA |
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| Source name |
resection
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| Organism |
Homo sapiens |
| Characteristics |
patient diagnosis: PDAC
tissue: resection
age: 71
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| Extracted molecule |
total RNA |
| Extraction protocol |
Live immune cells were sorted by EpCAM- /CD45+ (Biolegend, # clone 2D1 and 9C4) on a Sony SH800 cell sorter, stained with AO/PI (acridine orange / propidium iodide, Nexcelom ViaStain), and checked for viability > 80% using a Countess FL II Automated Cell Counter.
Gene expression (GEX) and V(D)J BCR libraries were constructed according to the manufacturer’s instructions (Single Cell 5’ Gene Expression V1 protocol, 10x Genomics). Libraries were sequenced on a combination of Illumina NextSeq500 (for GEX and V(D)J) and MiSeq (for V(D)J) flow cells.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic single cell |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
single-cell 5' Gene Expression
10x Genomics
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| Data processing |
Sequencing data was demultiplexed, mapped, and processed into gene/cell expression matrices using 10X Genomics' Cell Ranger software v6.0.0 (https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/what-is-cell-ranger). Gene expression reads were aligned to the human reference genome version gex-GRCh38-2020-A, while V(D)J reads were mapped to vdj-GRCh38-alts-ensembl-7.0.0, both available from the 10X Genomics website. Matrices from each separate sample were aggregated without read-depth normalization using the Cell Ranger aggr (v6.0.0) pipeline using the “normalize=None”.
Secondary analysis was carried out primarily using Loupe Browser (10X Genomics). Dead cells (% mitochondrial reads > 10%) and cells with the number of genes expressed less than 200 were also filtered out. Doublets were removed using a R package ‘Scrublet’. ScRNA-Seq Data was normalized, logarithm transformed and scaled. Principle component analysis (PCA) was run with 50 components using the top 4000 genes of each sample. The nearest neighbor algorithm was run with 50 PCAs and clustered in UMAP projections using Leiden clustering. All additional analyses were performed using Loupe Browser (10X Genomics) and the Python toolkit Scanpy. Immune cells clusters were defined using the set of markers and UMAP clusters: T cells (CD3+, TCR+, NCAM-), NK cells (CD3-, NCAM+), B cells (CD20+, CD79A+, IgKC low), PCs (JChainhi, IgKChi), myeloid cells (CD14+, CSF1R+), mast cells (CPA3+, GATA2+), fibroblasts (FAP+, ACTA2+, PDGFRA1+), epithelial cells (CK19+, CK18+, CK5+, CK14+).
Assembly: GRCh38
Supplementary files format and content: Gene expression: Single cell 5' cell/gene matrices in .mtx format generated by Cell Ranger for individual samples and for all samples aggregated. V(D)J analysis: annotated contig files in .csv format and assembled contig sequences in FASTA format for all detected BCR sequences output by cellranger vdj.
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| Submission date |
Jul 24, 2023 |
| Last update date |
Jul 31, 2023 |
| Contact name |
Jonathan Preall |
| E-mail(s) |
[email protected]
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| Phone |
5164224086
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| Organization name |
Cold Spring Harbor Laboratory
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| Lab |
Preall
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| Street address |
500 Sunnyside Blvd
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| City |
Woodbury |
| State/province |
NEW YORK |
| ZIP/Postal code |
11797 |
| Country |
USA |
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| Platform ID |
GPL18573 |
| Series (1) |
| GSE238163 |
Plasma cells in human pancreatic ductal adenocarcinoma secrete antibodies to widely expressed self-antigens |
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| Relations |
| BioSample |
SAMN36699180 |
| SRA |
SRX21145238 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7659637_Pt-19_GEX_barcodes.tsv.gz |
1.1 Mb |
(ftp)(http) |
TSV |
| GSM7659637_Pt-19_GEX_features.tsv.gz |
287.6 Kb |
(ftp)(http) |
TSV |
| GSM7659637_Pt-19_GEX_matrix.mtx.gz |
45.5 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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