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Sample GSM5979648

Query DataSets for GSM5979648

Status

Public on Mar 01, 2024

Title

surface protein library_Vax B_CD45+CD3- cells

Sample type

SRA

Source name

TC-1 mouse tumor infiltrated immune cell

Organism

Mus musculus

Characteristics

strain: C57BL/6
tissue: tumor
treatment: Vaccine

Treatment protocol

A total of 1.5x105 TC-1 were inoculated subcutaneously into right dorsal flanks of the mice in 100 μl phosphate buffered saline (PBS). Tumor-bearing mice were randomly grouped into treatment groups when tumors grew to around 30-60mm3. For E7-vaccine treatment, 0.5 μg of peptide and 30 μg of PC7a was mixed in 100 μl of PBS, subcutaneously injected on day 6-9 post tumor inoculation. For cGAMP treatment, the antibody was diluted in 200 μl of PBS and intraperitoneally injected on day 6, 9, 12 post tumor inoculation.

Extracted molecule

total RNA

Extraction protocol

TC-1 tumors harvested at day 17, 22 and day 27 post inoculation. Tumor tissues were cut into approximately 1 mm3 pieces in the RPMI-1640 medium (Invitrogen) with 2% fetal bovine serum (FBS), and enzymatically digested with MACS tumor Dissociation Kit (Miltenyi Biotec) for 40 min on gentleMACS™ Dissociator according to manufacturer’s instruction. The dissociated cells were subsequently passed through a 70 μm cell strainer and centrifuged at 500 g for 10 min.
As described in Single Cell V(D)J Reagent Kits with Feature Barcode technology for Cell Surface Protein User maual (10x Genomics, CG000186).

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NextSeq 500

Description

A8_SB45
B45_matrix.mtx.gz
B45_barcodes.tsv.gz
B45_features.tsv.gz

Data processing

The Cell Ranger (4.0.0) Software Suite was used to process the raw sequence data into gene expression profiles. The fastq files for each sample were processed with Cell Ranger count, which was used to align to mm10-2020-A reference genome, filter and quantify.
The output files of Cell Ranger for each sample included surface protein and gene expression (in UMI count). Merged surface protein and gene expression data were obtained using the cellranger aggr, which performed an inter-sample normalization and merged the results of all samples into one file, to remove the potential technique variability of samples.
For scTCR-seq data, TCR reads were aligned to the vdj_GRCm38_alts_ensembl-4.0.0 reference genome and TCR annotation was performed using the 10x cellranger vdj pipeline.
The Cd45 expression was defined as the average UMI counts of Ptprc, and those cells with Ptprc expression > 0 were kept for subsequent analysis. The Cd3 expression was defined as the average UMI counts of Cd3d, Cd3e, Cd3g, and those cells with Cd3 expression > 0 were kept for subsequent analysis.
The Seurat pipeline was applied to the combined expression data. Principle component analysis (PCA), t-distributed stochastic neighbor embedding (t-SNE), and Uniform Manifold Approximation and Projection (UMAP) were was performed on the scaled data of all genes.
Clusters were identified using shared nearest neighbor (SNN)-based clustering based on the same principal components. We used the function FindClusters for clustering with resolution from 0.1 to 1.5. For each resolution, the silhouette values were calculated. Such value was used to determine the optimaloptional cluster number k.
Assembly: mm10-2020-A
Supplementary files format and content: matrix.mtx file, features.tsv file, and a barcodes.tsv file with rawcounts from CellRanger Count

Submission date

Mar 28, 2022

Last update date

Mar 01, 2024

Contact name

Jiahui Chen

E-mail(s)

sophiacjh2020@gmail.com

Organization name

UTSW

Department

SCCC