GSM8683239: LC_20230323_synovium; Homo sapiens; RNA-Seq
The specimens used in this study were obtained from the Third Affiliated Hospital of Guangzhou University of Chinese Medicine (PJ-XS-20241029-001). Patients were diagnosed with hemophilia A and confirmed with HA through MRI. Fresh synovial membranes were collected from their knees after TKA, immersed in tissue storage solution (130-100-008, MACS).  Additionally, specimens from patients with OA and RA were similarly diagnosed through medical history and imaging, and fresh synovial membranes was obtained during TKA. The Chromium™ Single Cell 3' Solution is built on the GemCode technology microfluidics platform, which encapsulates gel beads with barcodes and primers along with individual cells in oil droplets to form GEMs (Gel Bead in Emulsion). GEM refers to the mixture of oil droplets containing barcoded Gel beads and cells, along with reaction reagents. The primers are composed of the full-length Illumina TruSeq Read 1 sequencing primer, a 16bp 10X Barcode sequence (used for cell identification), a 12bp unique molecular identifier (UMI) (used to distinguish different transcripts from the same cell and to remove PCR duplicates), and a 30 bp poly dT reverse transcription primer.The prepared cell suspension, 10X Barcode gel beads, and oil droplets are separately introduced into different channels (a total of 8 channels) of the Chromium Next GEM Chip G. The GEMs (Gel Bead in Emulsion) are formed through a microfluidic system. To achieve a single-cell reaction system, the concentration of the cell suspension is controlled between 700-1200 cells per microliter (µl). As a result, 90-99% of the GEMs do not contain any cells, while the majority of the remaining GEMs contain a single cell.Within the GEM (Gel Bead in Emulsion), the gel beads dissolve, and the cells lyse to release mRNA.  This mRNA is then reverse transcribed to produce barcoded cDNA for sequencing.  The liquid oil layer is disrupted, and cDNA amplification is carried out.  After purification, quality control is performed, and once the quality is confirmed, library construction is initiated.Enzymatic cutting is performed, followed by end-repairing of the fragments and the addition of an A-base overhang. Adapters are then ligated. The target-sized fragments are recovered using a size-selection kit, and the library is subjected to quality control and quantification.The constructed library structure, from 5' to 3', consists of: Illumina P5 adapter, P5 sequencing primer (black Read1 position), 16bp Barcode, 12bp Unique Molecular Identifier (UMI), poly(dT) random nucleotide (VN) sequence, inserted cDNA fragment, P7 sequencing primer (black Read2 position), sample Index (i7 index read), and Illumina P7 adapter. The可用 (yòng, which means 'available' or 'usable' in this context) sequence length for Read 1 is 28 base pairs (bp) (16bp Barcode + 12bp UMI), which is used to distinguish between cells and the different transcripts (mRNA) within each cell. The可用 (yòng) sequence length for Read 2 is 91 base pairs (bp), which is used to map to a reference genome to determine gene information.