GSM7073710: 395_IIR-KO; Homo sapiens; RNA-Seq
Stage 6 SC-islets were washed with PBS and dissociated with TrypLE Express for 15 min at 37°C. Cells were dissociated by pipetting up and down. Cell were then washed with warm DMEM media, resuspended in FACS-buffer (2% FCS, 2mM EDTA in PBS) and incubated with SYTOX-Blue for 5 min at room temperature. Live cells were then isolated by FACS and collected in 2% FCS in PBS. Subsequently, sorted cells were washed once with 2% FCS in PBS and cell numbers were determined using a hemocytometer. Approximately 16500 cells were loaded on one chip lane. Library was performed according to the manufacter's instructions (single cell 3' v3.1 protocol, 10x Genomics). Briefly, SC-islet 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.