title: GSM8152376: Day 5 of reprogrammed chondrocytes; Homo sapiens; RNA-Seq
library_construction_protocol: Chondrocytes collected at each time course were labeled with a 10X Genomics 3' CellPlex kit. After the cellular labeling, all cells were mixed, and cell sorting was performed on a FACSAria III Cell Sorter (BD Biosciences, San Diego, CA, USA) to remove dead cells and debris via forward scatter (FSC), side scatter (SSC), and 4',6-diamidino-2-phenylindole (DAPI)/Calcein-AM staining. Cells were then processed with Chromium Next GEM Single Cell 3ʹ GEM kit v.3.1 (10X Genomics Inc., Pleasanton, CA, USA) according to the manufacturer's instructions. Briefly, gel bead-in-emulsions (GEMs) were generated by loading gel beads, a master mix containing cell suspension, and partitioning oil onto a chip. Within each GEM, poly-A RNA from cell lysates was retrotranscripted into cDNA, incorporating an Illumina R1 primer sequence, a Unique Molecular Identifier (UMI), and a 10x Barcode specific to each cell. After GEM generation, the emulsions were broken, and the barcoded cDNA was cleaned using Silane DynaBeads. Subsequently, cDNA is amplified by PCR, incorporating P5 and P7 sequences essential for Illumina sequencing, ensuring that each cDNA molecule contains both the necessary elements for sequencing (Illumina R2 sequence, P5, and P7) and a sample index for multiplexing. The appropriate-sized fragments are selected using SPRIselect reagent for final library construction. This meticulous process ensures each cDNA fragment is equipped for high-throughput sequencing, maintaining the integrity and specificity of the single-cell RNA profiling.