GSM9821029: NMOs_Control_1; Homo sapiens; RNA-Seq
Twenty neuromuscular organoids (NMOs) from each condition were randomly pooled and dissociated into single-cell suspensions based on previously published protocols. Briefly, organoids were washed with PBS and incubated in Versene at 37◦C for 10–20 min, followed by gentle trituration to obtain single cells. DNase I was included during dissociation to reduce cell clumping. Cell suspensions were filtered through a 40-μm cell strainer and viability was assessed by trypan blue exclusion, consistently exceeding 95%. Cells were resuspended in PBS containing 0.04% BSA at approximately 700 cells/μL and processed for library preparation using the Chromium Single Cell platform (10x Genomics) according to the manufacturer's instructions. Single-cell RNA sequencing was performed using three independent biological replicates per condition. 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 appropriated 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.