GSM6705375: PDA-OVA_C2 scRNA seq; Mus musculus; RNA-Seq
Tumors were harvested, minced into smaller pieces with a scalpel and digested using the human tumor dissociation kit with gentleMACS Octo tissue dissociator (Miltenyi) according to the manufacturer's instructions (program '37C_h_TDK_3'). Following 1 h digestion, tumor cell suspensions were poured through a 100 μm strainer pre-coated with 2.5% BSA/PBS. Next, cells were washed twice over a 70 µm cell strainer and frozen in liquid nitrogen in freezing media (fetal bovine serum + 10% DMSO) until processing for single-cell RNA sequencing. Library was performed according to the manufacter's instructions (10x Genomics, v1 chemistry according to the manufacturer's protocol (#CG000086 Rev K)). 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. For gene expression library construction, 50 ng mass of sample were fragmented, end-repaired and A-tailed. For V(D)J library construction, cDNA was first enriched using T-cell mixes 1 by PCR amplification, followed by SPRIselect reagent size selection. Then, T-cell mix 2 was used for further enrichment by PCR amplification, followed by SPRIselect reagent size selection. Post target enrichment, quality control was checked on Agilent Bioanalyzer High Sensitivity Chip and quantified using Qubit Fluorometer and Qubit dsDNA HS Assay kit. For V(D)J library construction, 50 ng mass of sample were fragmented, end-repaired and A-tailed. During the library construction Ilumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and a sample index were added.