GSM9468566: Liver portal messenchymal cells from WT 014; Mus musculus; RNA-Seq
The bilio‐vascular tree is isolated from WT mouse liver by an enzyme digestion-based protocol. Liver is perfused with a mixture of collagenase in vivo and then explanted and further processed. A series of filter is then applied to collect portal tract cells. After cell isolation, we sort cells by FACS to collect lineage negative cells (gated as epithelial cell adhesion molecule Epcam-, CD31-, CD45-, CD11b- cells), to enrich mesenchymal cells. Library was performed according to the manufacter's instructions (single cell 3' v2 protocol, 10x Genomics). Briefly, messenchymal cells were resuspended in the master mix and loadedtogether 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 lumina R1 primer sequence, UniqueMolecular 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 lumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and a sample index were added.