GSM7429909: 19040X1 RFP-iPSCs-orgs 1; Homo sapiens; RNA-Seq
SNR-derived organoids and chimeras were rinsed with sterile Dulbecco's phosphate-buffered saline (DPBS, 1X) and chopped in small pieces using a scalpel. Organoids' pieces were collected in 1.5 mL tubes in DPBS and centrifuged 573 for 2 minutes at 1000 rpm. Samples were incubated in pre-warmed papain (Worthington Biochemical) containing 5% DNaseI (Sigma) at 37oC for 50 minutes. After incubation, cells were resuspended in Trypsin Inhibitor (Sigma) and manually triturated by pipetting up and down using P1000 pipette. Cells were centrifuged for 5 minutes at 1000 rpm before the next step. Cells were resuspended in cold DPBS containing 0.04% BSA. Samples were kept on ice from this point forward. Samples were quickly transferred to the High Throughput Genomics Core Facility at the University of Utah for cell viability assessment. Library was performed according to the manufacter's instructions (single cell 3' v2 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.