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| Status |
Public on Nov 16, 2023 |
| Title |
PBS,scRNAseq |
| Sample type |
SRA |
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| Source name |
Py8119 tumor
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| Organism |
Mus musculus |
| Characteristics |
cell type: Immune cells
tissue: Py8119 tumor
strain: C57BL/6
age: 6-12 weeks old
treatment: PBS
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| Extracted molecule |
total RNA |
| Extraction protocol |
Mice injected with VHHs phage display specific libraries for CD8 T cells, DC, and CD45 we also injected PBS as a negative control, and insertless phage as a positive control(three mice per group). After two hours tissues were mechanically minced and digested for 40 min with 100 U/ml Collagenase IV (Worthington), 10 mg/ml DNAse (Sigma Aldrich) and 10.5 mM Y-27632 (Sigma Aldrich) in HBSS (Corning) and RPMI 1640 (Mediatech, Inc., Corning) complemented with 3 % FetalClone II (HyClone). Immune cells were enriched using CD45 magnetic microbeads. Viability and cells count was measured by 0.4 % Trypan blue exclusion method. Cells were frozen in FBS + 10% DMSO. At the time of sequencing, thawed cells were counted using a TC20 automated cell counter (BioRad) and adjusted to 1000 cells/ul in 0.04 % BSA/PBS and 1 ml/sample were submitted to the Next Generation Sequencing Core.
Library was performed according to the manufacter’s instructions (single cell 3’ v3 protocol, 10x Genomics). Briefly, GCs were resuspended in the master mix and loaded together with partitioning oil and gel beads into the chip to generate the gel bead-in-emulsion (GEM). The poly-A RNA from the cell lysate contained in every single GEM was retrotranscripted to cDNA, which contains an Ilumina R1 primer sequence, Unique Molecular Identifier (UMI) and the 10x Barcode. The pooled barcoded cDNA was then cleaned up with Silane DynaBeads, amplified by PCR and the apropiated sized fragments were selected with SPRIselect reagent for subsequent library construction. During the library construction Ilumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and a sample index were added.
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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 |
10x Genomics
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| Data processing |
The demultiplexing, barcoded processing, gene counting and aggregation were made using the Cell Ranger software v5.0.1 (https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/what-is-cell-ranger)
Assembly: mm10
Supplementary files format and content: Hierarchical Data Formats HDF5 and matrix files
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| Submission date |
Jan 21, 2023 |
| Last update date |
Nov 16, 2023 |
| Contact name |
Todd Aguilera |
| E-mail(s) |
todd.aguilera@utsouthwestern.edu
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| Phone |
214-648-8935
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| Organization name |
UTSouthwestern- Medical Center
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| Department |
Molecular radiation biology
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| Lab |
Todd A. Aguilera
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| Street address |
2280 Inwood Rd
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| City |
Dallas |
| State/province |
TX |
| ZIP/Postal code |
75390 |
| Country |
USA |
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| Platform ID |
GPL24247 |
| Series (1) |
| GSE223428 |
Simultaneous selection of nanobodies for accessible epitopes on immune cells in the tumor microenvironment |
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| Relations |
| BioSample |
SAMN32859876 |
| SRA |
SRX19133526 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM6951234_PBS_filtered_feature_barcode_matrix.h5 |
9.5 Mb |
(ftp)(http) |
H5 |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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