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Sample GSM7659632 Query DataSets for GSM7659632
Status Public on Jul 31, 2023
Title Pt-20_BCR
Sample type SRA
 
Source name resection
Organism Homo sapiens
Characteristics patient diagnosis: PDAC
tissue: resection
age: 67
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.
 
Library strategy OTHER
Library source transcriptomic
Library selection other
Instrument model Illumina MiSeq
 
Description single-cell BCR
reads also produced on Illumina NextSeq 500
10x Genomics
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.
Library strategy: scBCR-seq
 
Submission date Jul 24, 2023
Last update date Jul 31, 2023
Contact name Jonathan Preall
E-mail(s) [email protected]
Phone 5164224086
Organization name Cold Spring Harbor Laboratory
Lab Preall
Street address 500 Sunnyside Blvd
City Woodbury
State/province NEW YORK
ZIP/Postal code 11797
Country USA
 
Platform ID GPL15520
Series (1)
GSE238163 Plasma cells in human pancreatic ductal adenocarcinoma secrete antibodies to widely expressed self-antigens
Relations
BioSample SAMN36699185
SRA SRX21145233

Supplementary file Size Download File type/resource
GSM7659632_Pt-20_all_contig.fasta.gz 7.3 Mb (ftp)(http) FASTA
GSM7659632_Pt-20_all_contig_annotations.csv.gz 3.6 Mb (ftp)(http) CSV
GSM7659632_Pt-20_filtered_contig.fasta.gz 157.1 Kb (ftp)(http) FASTA
GSM7659632_Pt-20_filtered_contig_annotations.csv.gz 227.1 Kb (ftp)(http) CSV
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file
Processed data are available on Series record

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