GSM6734885: SOC, replicate 4, scRNAseq; Homo sapiens; RNA-Seq
Freshly collected 18g core biopsy of liver allograft tissue obtained 12 months post-transplant from 4 STUDY and 4 SOC living donor liver transplant patients was minced into small pieces and incubated with 1 mg/ml collagenase digestion solution at 37 °C for 8 minutes. The isolated liver cell suspensions were resuspended in 20%∆ FCS, 2mM EDTA, 1XPBS solution. Cells were washed and re-suspended in 4% Ultrapure BSA/1x DPBS Library was performed according to the manufacter's instructions (single cell 3' v3 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.