|
| Status |
Public on Oct 29, 2020 |
| Title |
P91T-E |
| Sample type |
SRA |
| |
|
| Source name |
Tumor tissue of esophageal carcinoma
|
| Organism |
Homo sapiens |
| Characteristics |
patient: P91
tissue: esophageal carcinoma
cell population: CD45-
info: 10X 5' scRNA-Seq for CD45- cells from P91 TUMOR
|
| Treatment protocol |
Fresh ESCC tumors and their adjacent normal tissue samples were placed in RPMI-1640 medium (Invitrogen) with 20% fetal bovine serum (FBS; GE Healthcare Life Sciences) on ice immediately after surgical resection. Tissue sample processing was completed in 10 hours after collection. A portion of sample was cryosectioned, hematoxylin-eosin (H&E) stained and microscopically examined to assure that tumor sample contains > 40% cancer cells and normal tissue contains no cancer cells.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Fresh ESCC tumors and adjacent normal tissues were rinsed with PBS, gently cut into small pieces on ice and digested in RPMI-1640 medium (Invitrogen) containing 2 mg/ml collagenase IV (Gibco) and 0.5 mg/ml hyaluronidase (Sigma Aldrich) for 1 hour at 37oC. The digested cell suspension was subsequently filtered through a 70-μm cell strainer (BD Biosciences) and incubated in 1x red blood cell lysis buffer (BD Biosciences) on ice for 5 min. The remaining cells were suspended in 50 μl of PBS containing 1% FBS after washing once with the same medium. Single-cell suspension was stained with CD45-FITC (BD Biosciences) and sorted into immune (CD45+) or non-immune (CD45-) cells using a FACSAria flow cytometer (BD Biosciences). Sorted cells were examined and counted before subject to 10x Genomics Chips. We targeted for 7,000 cells recovered from each channel and used Chromium Single Cell 5’ Reagent Kits (10x Genomics) to prepared whole transcriptome RNA-sequencing libraries.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Description |
Raw data is being made available at https://bigd.big.ac.cn/gsa-human/ under the accession ID of HRA000195
|
| Data processing |
We processed the scRNA-seq data using Cell Ranger Single-Cell Software Suite (10x Genomics, version 2.1.0) with default parameters, aligned to the GRCh38 reference genome and the raw gene expression matrices were generated for each sample.
The expression matrixes of all samples were combined into CD45+ and CD45- tables. The combined tables were imported into the Seurat (version 2.3.4) toolkit for quality filtering and downstream analysis. For quality filtering, we first removed genes that were detected in < 0.1% of all cells and then filtered out cells with less than 500 detected genes or > 20% mitochondrial RNA content.
Genome_build: hg38
Supplementary_files_format_and_content: tab-delimited text files include UMI counts for each gene and each cell barcode (CD45xxx_UMIs.txt.gz)
Supplementary_files_format_and_content: tab-delimited text files including cell name, sample and annotated cell type for each cell (cellinfo.CD45xxx.txt)
|
| |
|
| Submission date |
Oct 28, 2020 |
| Last update date |
Jul 14, 2021 |
| Contact name |
Xiannian Zhang |
| E-mail(s) |
xiannian@ccmu.edu.cn
|
| Organization name |
Captital Medical University
|
| Department |
Department of neurology
|
| Street address |
10 YouAnMen Xitoutiao, Fengtai District
|
| City |
Beijing |
| ZIP/Postal code |
100086 |
| Country |
China |
| |
|
| Platform ID |
GPL24676 |
| Series (1) |
| GSE160269 |
Dissecting esophageal squamous-cell carcinoma ecosystem by single-cell transcriptomic analysis |
|
| Relations |
| BioSample |
SAMN16579669 |
| SRA |
SRX11403372 |
