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| Status |
Public on Jun 30, 2022 |
| Title |
Sample_04_tumor |
| Sample type |
SRA |
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| Source name |
intrahepatic tissue
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| Organism |
Homo sapiens |
| Characteristics |
cell type: CD45+ cells
tissue: intrahepatic tissue
tissue type: tumoral tissue
disease state: intrahepatic cholangiocarcinoma
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| Treatment protocol |
not applicable
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| Growth protocol |
not applicable
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| Extracted molecule |
total RNA |
| Extraction protocol |
Tissue was collected in 5ml of RPMI media. Further disaggregation of tissue into a single-cell solution for sequencing was completed using the MACs tumor dissociation kit (Miltenyi Biotec) with the standard tough tumor protocol. Briefly, the MACs tumor dissociation kit enzyme mix (300μl) was added to each sample. Next, samples were put into the gentleMACs Dissociator and ran through the tough tumor program. The cell suspension was then applied to a 70um cell strainer. Cells were pelleted, counted and frozen according to the slow freezing procedure in standard cryo-vials (1 ml cell suspension in the cryopreservation medium). Next, the cells were thawed, washed in PBS, stained with Live/dead Aqua Fluorescent Reactive Dye (Life Technologies) and anti-mouse CD45 antibody (30-F11, BD Biosciences) at 4°C for 20 minutes and sorted on a FACS Aria III (BD Biosciences) with a 100µm nozzle. Purity of live CD45+/CD45- cells was assessed after sorting, prior to downstream use.
CD45+/CD45- cells were resuspended in 1ml PBS plus 0.04% BSA and washed two times by centrifugation at 450 rcf for 7min. After the second wash cells were resuspended in 30 ul and counted with an automatic cell counter (Countess II, Thermo Fisher) to get a precise estimation of total number of cells recovered. Afterwards CD45+/CD45- cells of each sample were loaded into one channel of the Single Cell Chip A using the Single Cell 3’ v2 single cell reagent kit (10X Genomics) for Gel bead Emulsion generation into the Chromium system. Following capture and lysis, cDNA was synthesized and amplified for 14 cycles following the manufacturer’s protocol (10X Genomics). 50 ng of the amplified cDNA were then used for each sample to construct Illumina sequencing libraries. Sequencing was performed on the NextSeq500 Illumina sequencing platform following 10x Genomics instruction for reads generation.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
NextSeq 550 |
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| Description |
Lib_ALL_T-05_2
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| Data processing |
Raw blc files were converted into fastq files using the Cell Ranger pipeline suite of 10X Genomics (version 3.0.1).
The CellRanger analysis pipeline was used to generate a digital gene expression matrix starting from raw data.
Gene expression matrices were used as input for Seurat R package (R v3.6.1; Seurat v3.0.3)
Genome_build: Pre-build Human genome (version GRCh38) was used as genome reference and was provided as part of the CellRanger pipeline as a compatible transcriptome reference.
Supplementary_files_format_and_content: UMI counts per gene per cell
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| Submission date |
Apr 12, 2021 |
| Last update date |
Jun 30, 2022 |
| Contact name |
Alberto Termanini |
| E-mail(s) |
[email protected]
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| Organization name |
Istituto Clinico Humanitas
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| Lab |
Bioinformatic Unit
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| Street address |
Via Manzoni, 113
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| City |
Rozzano |
| State/province |
MI |
| ZIP/Postal code |
20089 |
| Country |
Italy |
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| Platform ID |
GPL21697 |
| Series (2) |
| GSE171899 |
High-dimensional single cell analysis of T cells infiltrating human intrahepatic cholangiocarcinoma [scRNA-Seq] |
| GSE171900 |
Hyperactivated Tregs abundantly infiltrate human intrahepatic cholangiocarcinoma and are controlled by transcription factor MEOX1 |
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| Relations |
| BioSample |
SAMN18715233 |
| SRA |
SRX10579816 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5237048_Sample_04_tumor_barcodes.tsv.gz |
23.7 Kb |
(ftp)(http) |
TSV |
| GSM5237048_Sample_04_tumor_features.tsv.gz |
297.6 Kb |
(ftp)(http) |
TSV |
| GSM5237048_Sample_04_tumor_matrix.mtx.gz |
13.2 Mb |
(ftp)(http) |
MTX |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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