title: GSM4420308: Day 6 DSP-GFP; Homo sapiens; RNA-Seq
library_construction_protocol: To dissociate WA25 and DSP-GFP into single cells, randomly selected day-6 organoids (n = 10) and day-29 organoids (n = 5) were pooled from each group. For day-48 WA25 cell aggregates, six representative skin organoids were collected, and the tail structure containing non-skin associated mesenchymal and neuronal cells was removed using two tungsten needles prior to dissociation in order to bias analysis toward skin tissue. Briefly, collected organoids were incubated with pre-warmed (37°C) TrypLE™ for total 8-30 min (depending on the organoid sizes and densities) in a 37°C incubator on a shaker at 65 rpm for a gentle swirl. During the incubation, the dissociation mixture was agitated every 3-10 min with gentle pipetting using wide-bore p1000 and p200 tips (Mettler-Toledo). By total 8-30 min of incubation, the tissue structure dissociated into a single cell suspension with no visible cell aggregation. Cold 3% BSA solution (Sigma-Aldrich) was added to the dissociated cell suspension in order to inactivate TrypLE enzymatic activity. The suspension was filtered through a 40 µm Flowmi™ cell strainer (Bel-Art™) in order to eliminate any debris. An additional three washes with cold 3% BSA solution were performed. Cells were resuspended in 3% BSA, and filtered through a 40 µm Flowmi™ cell strainer. Viability (live cell percentage) and live cell count was determined using Trypan Blue (used at 1:1 ratio) and Countess II. Final cell concentration was ~1,000 cells/µl and >90% viability. Single cell 3' RNA-seq experiments were conducted using the Chromium single cell system (10x Genomics, Inc) and the NexSeq500 or NovaSeq 6000 sequencer (Illumina, Inc). Approximately 17,000 cells (targeting for 10, 000 cells) per sample were added to a single cell master mix, following the Chromium Single Cell 3' Reagent Kits v2 User Guide, CG00052 Ver B (10x Genomics, Inc). Day-6 samples were processed using the Version 3 Chromium Single Cell 3' Reagent Kits. Along with the single-cell gel beads and partitioning oil in separate wells of a Single Cell A Chip, the single cell reaction mixture was loaded to the Chromium Controller for GEM generation and barcoding, followed by cDNA synthesis and library preparation. At each step, the quality of cDNA and library was examined by Bioanalyzer. The resulting library was sequenced in a custom program for 26b plus 98b paired-end sequencing on an Illumina NextSeq500 or 28b plus 91b paired-end on Illumina NovaSeq 6000 to a read depth of >30,000 reads per cell.
attributes.tag: GEO Accession
attributes.value: GSM4420308