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Sample GSM6262665

Query DataSets for GSM6262665

Status

Public on Sep 30, 2022

Title

S39_D190103_N

Sample type

SRA

Source name

stomach

Organism

Homo sapiens

Characteristics

tissue: stomach
tissue type: Adjacent non-tumor normal Sample from individual D190103
treatment: none

Extracted molecule

polyA RNA

Extraction protocol

Tumors and adjacent non-cancer tissues were cut into approximately 1-2 mm3 pieces in the RPMI-1640 medium (Gibco) with 10% fetal bovine serum (FBS, GIBCO), and enzymatically digested with gentleMACS (Miltenyi) for 60 min on a rotor at 37°C, according to manufacturer’s instruction. The dissociated cells were subsequently passed through a 100 µm SmartStrainer and centrifuged at 400 g for 5 min. After the supernatant was removed, the pelleted cells were suspended in red blood cell lysis buffer (TIANDZ) and incubated on ice for 1-2 min to lyse red blood cells. After washing twice with 1× PBS (Gibco), the cell pellets were re-suspended in sorting buffer (PBS supplemented with 1% fetal bovine serum (FBS, Gibco)). Single cell suspensions were stained with antibodies against CD326 (EPCAM, BioLegend, Cat #324207) and 7-AAD (eBioscience, Cat# 00-6993-50) for FACS sorting, performed on a BD Aria SORP instrument. Expression levels of EPCAM and permeability of 7-AAD were gated by their negative controls of unstained cells and positive controls of beads stained by each antibody. Based on FACS analysis, single cells were sorted into 1.5 ml tubes (Eppendorf) and counted manually under the microscope. The concentration of single cell suspensions was adjusted to 500-1200 cells/ul.
Cells were loaded to the 10x Chromium Microfluidic Chips for single-cell RNA and TCR library preparation. All the subsequent steps were performed following the standard manufacturer’s protocols. Purified libraries were analyzed by an Illumina Hiseq-4000 sequencer with 150-bp paired-end reads.

Library strategy

RNA-Seq

Library source

transcriptomic single cell

Library selection

cDNA

Instrument model

Illumina HiSeq 4000

Description

scgex.txt.gz
metadata.txt.gz

Data processing

Cell Ranger 3.0.0 was used to quantify gene expression level and to identify TCR sequences.
A set of quality thresholds was applied to filter out low-quality cells, including detection of 200-7500 genes, 500-75000 UMI counts, and less than 10% mitochondrial reads.
UMI counts were then normalized using SCTransform with default parameters.
Assembly: GRCh38
Supplementary files format and content: text files for expression matrices and cell metadata

Submission date

Jun 23, 2022

Last update date

Sep 30, 2022

Contact name

Zemin Zhang

E-mail(s)

zemin@pku.edu.cn

Organization name

Peking University

Street address

5 Yiheyuan Rd, Haidian