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| Status |
Public on May 11, 2023 |
| Title |
171: P17, colon, tumor, anti-PD-1, scRNA-seq |
| Sample type |
SRA |
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| Source name |
colon
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| Organism |
Homo sapiens |
| Characteristics |
subject: P17
cell type: Goblet cells
tissue: colon
genotype: tumor
treatment: anti-PD-1
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| Extracted molecule |
total RNA |
| Extraction protocol |
Tumor biopsy samples were first stored in liquid nitrogen with the SenotechTM Tissue Storage Buffer(#JZ-SC-5802，Senotech Genomics, Shanghai, China)or proceed directly to tissue disassociation. Heat the frozen tissue in a 37 ° C water bath,then transfer the tissue samples together with the freezed-storage solution to a new 15mL centrifuge tube, add 5mL preheated RPMI-1640 medium (Gibco).Centrifuge at room temperature at 300×g for 5min to remove the supernatant without disturbing the tissue sample at the bottom of the centrifuge tube.The tissue samples enzymatically digested with Neuronal Isolation Enzyme(Thermo Scientific) for 20-30 min at 37 C according to the manufacturer’s protocol.The dissociated cells were next passed through a 40-mm cell-strainer (BD) in the RPMI-1640 medium (Invitrogen) with 10% FBS until uniform cell suspensions were obtained. Subsequently, the suspended cells were passed through cell strainers and centrifuged at 400 g for 10 min. Red blood cells were removed via the same procedure described above. The recommended cell washing and resuspension solution is 1X PBS (calcium and magnesium free) containing 0.04% weight/volume BSA (400 µg/ml).
Single-cell RNA-seq libraries were prepared using the Chromium Next GEM Single Cell 3’ Kit v2 or v3 from 10x Genomics, following the manufacturer’s instructions. In brief, single cells were resuspended in PBS containing 0.04% BSA to a final concentration of 500~1,200 cells/ml as determined by Countess 3 Automated Cell Counters(Invitrogen). ~10,000 cells were captured in droplets to generate nanoliter-scale Gel bead in EMulsion (GEMs). GEMs were then reverse transcribed in a C1000 Touch Thermal Cycler (Bio-Rad) programmed at 53C for 45 min, 85C for 5 min, and held at 4C. After reverse transcription and cell barcoding, emulsions were broken and cDNA was isolated and purified with Cleanup Mix containing DynaBeads and SPRIselect reagent (Thermo Fisher Scientific), followed by PCR amplification. Amplified cDNA was fragmented and end-repaired, double-sided size-selected, PCR-amplified with sample indexing primers, and double-sided size-selected or RNA-seq library construction. Libraries prepared according to the manufacturer’s user guide were then purified and profiled for quality assessment. Single-cell RNA libraries were sequenced by an Illumina NovaSeq 6000 or BGISEQ DNBSEQ-T7 sequencer with 150 bp paired-end reads.
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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 vcellranger-5.0.1 (https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/what-is-cell-ranger)
Assembly: CRCh38
Supplementary files format and content: Tab-separated values files and matrix files
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| Submission date |
Jun 06, 2022 |
| Last update date |
May 11, 2023 |
| Contact name |
Cheng Wu |
| E-mail(s) |
wuch55@mail2.sysu.edu.cn
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| Phone |
18846145680
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| Organization name |
Sun Yat-sen University
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| Street address |
No. 74, Zhongshan 2nd Road, Yuexiu District, Guangzhou
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| City |
Guangzhou |
| ZIP/Postal code |
510600 |
| Country |
China |
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| Platform ID |
GPL24676 |
| Series (1) |
| GSE205506 |
Remodeling of the Immune and Stromal Cell Compartment by PD-1 Blockade in Mismatch Repair-Deficient Colorectal Cancer |
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| Supplementary file |
Size |
Download |
File type/resource |
| GSM6213963_Colon_171_v3_barcodes.tsv.gz |
43.3 Kb |
(ftp)(http) |
TSV |
| GSM6213963_Colon_171_v3_features.tsv.gz |
297.6 Kb |
(ftp)(http) |
TSV |
| GSM6213963_Colon_171_v3_matrix.mtx.gz |
14.2 Mb |
(ftp)(http) |
MTX |
| Raw data not provided for this record |
| Processed data provided as supplementary file |
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