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| Status |
Public on Mar 23, 2022 |
| Title |
KRC29 |
| Sample type |
SRA |
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| Source name |
KRC tumor
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| Organism |
Mus musculus |
| Characteristics |
tissue: KRC tumor
cell type: digested pancreas tumor
mouse strain: C57Bl/6 - DBA/2 mixed background
treatment: None
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| Extracted molecule |
total RNA |
| Extraction protocol |
Tumors and pancreas were enzymatically digested into a single-cell suspension: a 10× digestion buffer was prepared in PBS: collagenase type I (450 units/mL, Worthington Biochemical), collagenase type II (150 units/mL, Worthington Biochemical), collagenase type III (450 units/mL, Worthington Biochemical), collagenase type IV (450 units/mL, Gibco, Thermo Fisher Scientific), elastase (0.8 units/mL, Worthington Biochemical), hyaluronidase (300 units/mL, MilliporeSigma), and DNAse type I (250 units/mL, MilliporeSigma). Tumors and pancreas were enzymatically digested into a single-cell suspension. Briefly, freshly dissected tissue was placed into a 10-cm tissue culture dish, and a sterile razor blade was used to cut the tissue into fine pieces. Samples were resuspended in PBS and washed twice by centrifuge at 480 g for 3 minutes and added to a 50-mL tube containing 1× digestion buffer (Worthington Biochemical) containing 1% FBS. The tube was incubated on a shaker at 37°C for 60 minutes. Then 35 mL of PBS was added, and cells were washed 3 times before filtering out debris using a 70-μm mesh filter (MilliporeSigma). Single cells were resuspended in 100 μL of PBS in preparation for single-cell library creation.
Single cell suspensions were resuspended in PBS containing 0.04% w/v bovine serum albumin and brought to a concentration of 200–700 cells/μL. Appropriate volume of cells was loaded with single cell 5’ gel beads into a single cell chip and run on the Chromium Controller (10x Genomics, Inc; Pleasanton, CA). Dynabeads MyOne Silane magnetic beads (Thermo Fisher Scientific) were used to clean up the gel bead emulsion reaction mixture. Full-length, barcoded cDNA was amplified by PCR after cleanup. All samples were run on Agilent Tapestation 4200 using DNA high sensitivity D5000 tape. During library preparation, sample index and Illumina adapter sequences (Illumina Inc; San Diego, CA) were added. After library preparation quality control was performed using DNA D1000 tape on the Agilent Tapestation 4200 and final concentration was measured using the Qubit 4 Fluorometer DNA HS assay (Thermo Fisher Scientific). All samples were loaded at a concentration of 1.5 pM and run on the Illumina NextSeq500 High Output Flowcell. The run configuration was 26 base pairs (bp) × 98 bp × 8.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
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| Data processing |
Raw base call (BCL) files were demultiplexed and converted to FASTQ files, which were subsequently used to generate feature-barcode matrices using Cell Ranger RNA v3.1 (10x Genomics). PM sample was run through the Single cell RNA-seq app on BaseSpace. Briefly, reads were aligned against a reference genome using Spliced Transcripts Alignment to a Reference (STAR), followed by barcode tagging and BAM indexing. Count file was generated using gene UMI counter and with cells passing quality filter based on cells above background and passing knee filter.
Initial single-cell analysis was performed using Seurat v3. Cells containing less than 200 genes were removed, and log-normalized expression of 2,000 variable genes were used to reduce the data into two-dimensional space.
Cells from all 10 samples were combined using FindIntegrationAnchors and IntegrateData functions. Cell identities were manually assigned using highly expressed genes.
Genome_build: mm10
Supplementary_files_format_and_content: filtered_feature_bc_matrix
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| Submission date |
Nov 16, 2021 |
| Last update date |
Mar 23, 2022 |
| Contact name |
Anirban Maitra |
| E-mail(s) |
[email protected]
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| Organization name |
The University of Texas MD Anderson Cancer Center
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| Department |
Translational Molecular Pathology
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| Lab |
Maitra laboratory
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| Street address |
6565 MD Anderson Blvd
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| City |
Houston |
| State/province |
TX |
| ZIP/Postal code |
77030 |
| Country |
USA |
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| Platform ID |
GPL19057 |
| Series (1) |
| GSE188946 |
Single cell RNA sequencing profiles on whole tumors from a Kras/Rnf43 genetically engineered mouse model of pancreatic adenocarcinoma |
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| Relations |
| BioSample |
SAMN23181293 |
| SRA |
SRX13148772 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5692187_KRC29_filtered_feature_bc_matrix.tar.gz |
9.8 Mb |
(ftp)(http) |
TAR |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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