GSM8517320: WT,GEX; Mus musculus; RNA-Seq
Liver tissues enriched for metastatic B16F10 melanomas were harvested from WT or Vsig4-/- mice, cut into small pieces and digested with 0.5 mg/mL collagenase IV (Sigma) and 5 U/mL DNase I (Sigma) for 20 mins at 37℃ under shaking. The cell suspension was filtered through a 70 µm cell strainer and centrifugated at 400g for 5 mins at 4℃, followed by lysis of red blood cell inn ACK buffer (Biolegend).DAPI-CD45+ singlets were sorted by FACS and further processed for scRNA-seq. 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.Library was performed according to the manufacter's instructions (single cell 5' v1.1 protocol with feature barcoding and VDJ sequencing, 10x Genomics).