GSM9125363: Dark SkO; Homo sapiens; RNA-Seq
Skin organoid dissociation for scRNA-seq. Day-130 organoids (n = 1 per group) were incubated in 10% Dispase (Corning) containing the CEPT cocktail (hereafter, CEPT; final concentrations: 50 nM Chroman 1 (MedChem Express), 5 µM Emricasan (Selleck Chemicals), 1x Polyamine Supplement (Sigma-Aldrich), and 0.7 µM Trans-ISRIB (Tocris)) for 30 min at 37°C. During incubation, samples were gently pipetted every 10 minutes–initially using wide-orifice p1000 or p200 tips (Rainin), followed by standard p200 tips to enhance dissociation. Residual aggregate clumps were briefly centrifuged into pellets, and the dissociated cell suspensions were retained and kept on ice. The pellets were then resuspended in TrypLE™ (Gibco) supplemented with CEPT and further incubated until fully dissociated (typically an additional 30 min at 37°C with 10 min interval pipetting). Dispase and TrypLE™ fractions were pooled and gently mixed. Enzymatic activity was neutralized by adding 3% cold BSA (Invitrogen) in CEPT-supplemented medium at a 2:1 ratio volume ratio (medium:cell suspension). Cells were centrifuged into pellets (230 rcf, 5 min 30 sec, room temperature (RT)) and washed twice with a neutralizing buffer. The final suspension was filtered through a 40 µm Flowmi™ strainer (Bel-Art™). Cell viability and concentration (target: ~1,000 cells/µL; >90% viable) were determined using Trypan Blue and a Countess™ III FL Automated Cell Counter. Single-cell cDNA library construction and sequencing. Day-130 light- and dark-SkO samples were processed for single-cell transcriptomic profiling using the Chromium Single Cell 3' system (10x Genomics) and sequenced on NovaSeq 6000 platform. Approximately 17,000 cells per sample (targeting recovery of ~10,000 cells) were resuspended in the reverse transcription (RT) master mix from the Chromium Single Cell 3' Reagent Kit v3.1 (10x Genomics). Cell suspensions were then combined with barcoded gel beads and partitioning oil in a Single Cell A Chip and loaded into the Chromium Controller for generation of Gel Beads-in-Emulsion (GEMs). RT, cDNA amplification, and library preparation were performed according to the manufacturer's protocol (CG000183, Rev C). The quality of amplified cDNA and final libraries was assessed using a Bioanalyzer (Agilent). Sequencing was conducted with paired-end read lengths for the NovaSeq 6000–to achieve a read depth of >30,000 reads per cell.