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Sample GSM7719486

Query DataSets for GSM7719486

Status

Public on Oct 18, 2023

Title

EMT6 rep 2 [Champions Oncology]

Sample type

SRA

Source name

Tumor

Organism

Mus musculus

Characteristics

tissue: Tumor
cell line: EMT6
source: Champions Oncology

Extracted molecule

total RNA

Extraction protocol

Frozen samples of primary human lung tumors underwent dead cell removal and T cell enrichment using the Sony MA900 Cell Sorter with a DAPI live/dead cell discriminator and staining for CD45 and CD3. Following dead cell removal and T cell enrichment (CD45+ 972 CD3+), cell count and viability were assessed using acridine orange/propidium iodide on a Nexcelom Cellometer and cells were washed twice in PBS + 0.04% bovine serum albumin. Following successful isolation, 15,000 cells per sample were loaded into the 10X Chromium chip using the Chromium Single Cell (v3.1 chemistry) with the intention of targeting 10,000 cells/sample for sequencing.
3′ GEX scRNA-Seq libraries were prepared according to manufacturer recommendations. Libraries were quality controlled on the Agilent TapeStation. Libraries were pooled and sequenced on the NovaSeq 6000 S2 100 flow cell targeting an average depth of 25,000 reads per cell (×10,000 cells/sample) or 250 million reads/sample.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NovaSeq 6000

Description

tpm.all.final.txt

Data processing

FASTQ data files containing raw reads for each of the three samples were generated and analyzed separately by the vendor to generate single-cell feature (gene) counts using the function “cellranger count” from 10x Genomics Cell Ranger 5.0.0
A single feature count matrix aggregating across all three samples was generated using “cellranger aggr” from 10x Genomics Cell Ranger 6.0.0
From the resulting matrix, cells that had fewer than 1000 total counts; or more than 25% of their counts going to mitochondrial genes, or which had zero CD3 expression (zero counts for CD3D, CD3E, and CD3G), were excluded.
The reduced matrix was reanalyzed using “cellranger reanalyze”
Assembly: hg38
Supplementary files format and content: barcodes.tsv.gz is a compressed file containing the cell names for the scRNA-seq data.
Supplementary files format and content: features.tsv.gz is a compressed file containing the gene names for the scRNA-seq data.
Supplementary files format and content: matrix.mtx.gz is a compressed file containing the cell by gene count matrix for the scRNA-seq data.
FASTQ files from RNA-seq of all models were uniformly processed in-house using a pipeline consisting of adapter trimming using Cutadapt v1.16;
read alignment was performed using STAR v2.5.2b, to the mm10/GRCm38 genome and transcriptome annotated by GENCODE and downloaded from UCSC
Isoform and gene-level transcript quantification was conducted using RSEM v1.2.31
The FFPE samples for the in-house CT26 model were run using RSEM parameters for a TruSeq stranded library (--forward- prob=0)
As a normalization step, we perfomed a rescaling of the gene- and isofrom-level quantification TPMS to exclude nonprotein-coding genes, a step that we have founf to improve the clustering of samples with mixed library prepartaion methods
Assembly: mm10
Supplementary files format and content: tpm.all.final.txt is text file containing per-sample gene-level quantification, in units of TPM (transcripts per million reads). Columns are sample IDs, rows are genes, with a header row, and tab-separated fields.

Submission date

Aug 18, 2023

Last update date

Oct 18, 2023

Contact name

Alyson J Smith

Organization name

Seagen Inc.

Department

Research

Street address

21717 30th Drive S.E., Building 3