GSM7105039: LC-6; Mus musculus; RNA-Seq
FACS analysis of tumour-infiltrating lymphocytes and tumour-infiltrating lymphocytes isolated in tumour models was performed after staining with a PE-conjugated anti- mouse CD45 antibody (clone 30-F11, Biolegend). CD45+ immune cells were then enriched using a BD FACS Aria III flow cytometer. Cell viability was monitored in real time during the preparation of single CD45+ immune cell suspensions. Ten thousand cells (~600 single cells per microlitre) from each experimental group were barcoded and pooled using a 10x Genomics device. Samples were prepared according to the manufacturer's protocol and sequenced on an Illumina NextSeq sequencer. The Cell Ranger Analysis Pipeline (v.3.0.2) was used for sample demultiplexing, barcode processing, alignment, filtering, UMI counting, and aggregation of sequencing runs. For quality control of the single-cell RNA-sequencing process, cells with fewer than 300 genes detected and cells with mitochondrial-encoding gene transcript counts exceeding 15% of total transcript counts were removed from subsequent analyses. Genes detected in less than three cells in the entire dataset were also excluded, resulting in a preliminary expression matrix of 15603 cells. After obtaining the digital gene expression data matrix, dimensionality reduction, clustering and differential gene expression analysis were performed using Seurat (v.3.0.0.9000) Library was performed according to the manufacter's instructions (single cell 3' v2 protocol, 10x Genomics). Briefly, GCs were resuspended in the master mix and loaded together with partitioning oil and gel beads into the chip to generate the gel bead-in-emulsion (GEM). The poly-A RNA from the cell lysate contained in every single GEM was retrotranscripted to cDNA, which contains an Ilumina R1 primer sequence, Unique Molecular Identifier (UMI) and the 10x Barcode. The pooled barcoded cDNA was then cleaned up with Silane DynaBeads, amplified by PCR and the apropiated sized fragments were selected with SPRIselect reagent for subsequent library construction. During the library construction Ilumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and a sample index were added.