GSM7842976: Control Sample25 - Gene expression; Mus musculus; RNA-Seq
Liver metastases were isolated and dissociated into single cells by physical (chopping the tissue into small pieces) and enzymatic (collagenase, Dispase II and DNAse) digestion followed by a filtering step using a 0.4mm cell strainer. No antibody staining was performed, single cells were resuspended in autoMACS running buffer containing 7AAD (BioLegend/ 420404) in a 1/50 dilution. Viable cells were sorted based by gating on single 7AAD negative cells. Library was performed according to the manufacter's instructions (single cell 3' v3.1, 10x Genomics, ref. CG000204). Briefly, sorted cells 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.