GSM6533784: CTRL, brain organoid, day56 [scRNA-seq]; Homo sapiens; RNA-Seq
Three sets of human D56 forebrain organoids derived from three sets of CTRL and WS iPSCs were dissociated with 3 mL Tryp-LE (Sigma-Aldrich) and for 15 minutes at 37°C, followed by 2 washes with 3% FBS in DPBS and gentle titration using a P1000 pipette. A single-cell suspension was subsequently collected into 2 mL microtubes with a cell density of 1000 cells/μL. Then the suspensions were added to a 10X Genomics GemCode Single-cell instrument to generates a single-cell Gel Bead-In-EMlusion 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.