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| Status |
Public on Jul 08, 2024 |
| Title |
P20-N-II |
| Sample type |
SRA |
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| Source name |
Adjacent normal tissue
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| Organism |
Homo sapiens |
| Characteristics |
tissue: Adjacent normal tissue
treatment stage: On
patientid: P20
cancer type: CRC
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
For tissue samples, collected tumor or adjacent normal biopsies was stored in the tissue storage solution immediately after resection and transferred on ice. Samples were washed twice and cut into approximately 1-2 mm3 pieces before being collected in the RPMI-1640 medium (Gibco) with 10% fetal bovine serum (FBS, Gibco), and projected to enzymatical digestion with gentleMACS Tumor Dissociation Kit (Miltenyi Biotec) for 60 min on a rotor at 37°C according to the manufacturer’s protocol. The dissociated cells then passed through a 100-mm SmartStrainer in the RPMI-1640 medium (Invitrogen) with 10% FBS and centrifuged at 400 g for 5 min. After the removal of the supernatant, the pelleted cells were suspended in 1 mL of red blood cell lysis buffer (TIANDZ) and incubated on ice for 1 min for red blood cell lysis and quickly terminate the reaction with RPMI-1640 medium (Invitrogen, with 10% FBS). After being centrifuged at 400 g for another 5 min, the cell pellets would be re-suspended in sorting buffer (PBS supplemented with 1% FBS). Blood samples were collected in EDTA anticoagulant tubes and kept on ice for transferring. After restoring to room temperature and mixing thoroughly, peripheral blood mononuclear cells (PBMCs) were isolated using HISTOPAQUE-1077 (Sigma-Aldrich) solution. Briefly, peripheral blood was carefully layered onto equal volume HISTOPAQUE-1077 without breaking the plasma-HISTOPAQUE-1077 interface. After centrifugation at 400 g for 30 min, the lymphocyte layer was carefully transferred to a new tube and washed twice with 1x phosphate-buffered saline (PBS, Invitrogen). After red blood cells lysis following the aforementioned procedure, the collected lymphocytes were then re-suspended in sorting buffer (PBS supplemented with 1% FBS).
To obtain high-quality cell pools before subsequent library construction, single cell suspensions collected from blood or tissue samples were stained with antibodies against 7AAD (Thermo Fisher), CD235a (BioLegend) for FACS sorting (BD Aria III instrument). By gating 7AAD- CD235a- cells, living cells were enriched, and residuary red blood cells were excluded. Gated cells were sorted into 1.5 mL LoBind tubes (Eppendorf) and counted manually under the microscope. The concentration of single cell suspensions was adjusted to 500-1200 cells/l. 10,000~18,000 cells were loaded for the 10x Chromium Single cell 5’ and VDJ library construction (10x Genomics), with subsequent steps performed following the standard manufacturer’s protocols. Purified libraries were analyzed by an Illumina NovaSeq with 150-bp paired-end reads.
10x Chromium Single cell 5’ + VDJ Library
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| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina NovaSeq 6000 |
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| Data processing |
For 10X data, Cell Ranger 3.1.0 was used to quantify gene expression level and identify TCR sequences.
Assembly: GRCh38 for RNA expression
Supplementary files format and content: text files for expression matrices, cell metadata
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| Submission date |
Jul 05, 2023 |
| Last update date |
Jul 08, 2024 |
| Contact name |
Zemin Zhang |
| E-mail(s) |
zemin@pku.edu.cn
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| Organization name |
Peking University
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| Department |
BIOPIC
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| Lab |
Zemin Zhang's Lab
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| Street address |
Yiheyuan Road
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| City |
Beijing |
| State/province |
Beijing |
| ZIP/Postal code |
100091 |
| Country |
China |
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| Platform ID |
GPL24676 |
| Series (1) |
| GSE236581 |
Spatiotemporal single-cell analysis decodes cellular dynamics underlying different responses to immunotherapy in Colorectal Cancer |
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| Supplementary data files not provided |
| Raw data not provided for this record |
| Processed data are available on Series record |
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