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| Status |
Public on Sep 13, 2022 |
| Title |
PDAC5, scRNAseq |
| Sample type |
SRA |
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| Source name |
Pancreatic cancer
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| Organism |
Homo sapiens |
| Characteristics |
tissue: Pancreatic cancer
treatment: No treatment
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| Extracted molecule |
total RNA |
| Extraction protocol |
All fresh resection specimens were preserved in the tissue storage solution (Miltenyi, Cat. no. 130-100-008) on ice and transported to the laboratory in CapitalBio Technology company within 1.5-2 hours. To ensure the success of tissue dissociation, multiple resection specimens from the same patients were digested. All specimens were cut into around 1 mm pieces and incubated in an optimal digestive solution, including enzyme cocktail, consisting of the Type VIII Collagenase (Sigma-Aldrich, Cat. no. C2139), DNase I (Sigma-Aldrich, Cat. no. D5025), trypsin inhibitor (Sigma-Aldrich, Cat. no. T6522), and Dispase II (neutral protease, grade II, Sigma-Aldrich, Cat. no. 4942078001). The single cell suspension was filtered with a 40 μm cell strainer (BD, Cat. no. 352340), then incubated with red blood cell lysis buffer (Roche, Cat. no. 11814389001) at 4°C for 10 minutes.
The concentration of single cell suspension was determined using the Count Star instrument and adjusted to 1000 cells/μl. The cell suspension was loaded onto the Chromium single cell controller (10x Genomics) to generate single-cell gel beads in the emulsion using single cell 3’ Library and Gel Bead Kit v3.1 (10x Genomics, Cat. No. 1000121) and Chromium Next GEM Chip G Single Cell Kit (10x Genomics, Cat. No. 1000120) according to the manufacturer’s instructions. Briefly, cells were suspended in PBS containing 0.04% BSA. About 6,000 cells were loaded to each channel, and the target cell will be captured about 3000 cells per channel. Captured cells were lysed and the released RNA were barcoded by reverse transcription in each GEM. Reverse transcription was completed in 200 ul tubes (NEST Biotechnology, Cat. No. 401001) on a S1000TM Touch Thermal Cycler (Bio Rad) at 53°C for 45 minutes, then at 85°C for 5 min, and hold at 4°C. The cDNA libraries were generated, amplified, and quality assessed using the Agilent 4200. Finally, the cDNA libraries were sequenced using an Illumina Novaseq6000 sequencer with a sequencing depth of at least 100,000 reads per cell with paired-end 150 bp (PE150) reading strategy.
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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 |
Raw data (BCL files) from Illumina Novaseq6000 platform was converted to fastq files using Illumina-implemented software bcl2fastq (v2.19.0.316). cDNA reads were aligned to human reference genome (GRCh38). Low-quality cells and genes filtering, barcode and UMI counting were performed with the cellranger software (v6.1.2) to obtain the filtered gene-cell matrixes. Next, gene-cell matrixes were imported into R software to further filtered out low-quality cells (< 500 genes/cell, > 25% mitochondria genes, < 1000 transcripts/cell) and genes (<10 cells/gene) using ‘Seurat’ R package (v3.2.3). Gene expression levels were normalized (LogNormalize) with “NormalizedData” function. A total of 2,000 highly variable genes were selected and used to conduct PCA reduction dimension. The t-distributed stochastic neighbor embedding (t-SNE) was performed. The ‘Soupx’ R package was applied to reduce the ambient mRNA contamination. Doublets were identified using the ‘DoubletFinder’ R package (v2.0.3), assuming that it was around 5% doublet formation rate to the loaded cells per specimen in a droplet channel. In addition, the ‘Harmony’ R package (https://github.com/immunogenomics/harmony) was used to integrate gene-cell matrixes derived from different specimens. We identified each cell cluster by matching the cluster-specific genes with known signatures of cell populations reported in previous studies and CellMarker database.
Assembly: GRCh38
Supplementary files format and content: Tab-separated values files and matrix files
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| Submission date |
Sep 08, 2022 |
| Last update date |
Nov 29, 2022 |
| Contact name |
Kai Chen |
| E-mail(s) |
[email protected]
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| Phone |
+8618086411579
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| Organization name |
Peking University First Hospital
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| Department |
General Surgery
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| Street address |
8th Xishiku Street
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| City |
Beijing |
| ZIP/Postal code |
100034 |
| Country |
China |
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| Platform ID |
GPL24676 |
| Series (1) |
| GSE212966 |
Single-cell RNA-seq reveals immune landscape of pancreatic cancer |
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| Relations |
| BioSample |
SAMN30732553 |
| SRA |
SRX17495380 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM6567163_PDAC5_barcodes.tsv.gz |
21.7 Kb |
(ftp)(http) |
TSV |
| GSM6567163_PDAC5_genes.tsv.gz |
287.6 Kb |
(ftp)(http) |
TSV |
| GSM6567163_PDAC5_matrix.mtx.gz |
40.0 Mb |
(ftp)(http) |
MTX |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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