GSM8079184: endplate cells, patient 1, scRNAseq; Homo sapiens; RNA-Seq
The cartilage endplate tissue of the intervertebral discs resected during the surgery was transported immediately to the laboratory under 4°C cold storage for single-cell suspension preparation. The vertebral endplate tissues were washed twice with sterile PBS buffer to remove any blood and prevent contamination, left to stand, and the supernatants were aspirated. The tissues were cut quickly into small 1-2 mm pieces with pre-cooled ophthalmic scissors and placed on ice. And added pre-warmed digestive enzymes (collagenase, neutral protease and DNAase I), mixed gently with a pasteurizing pipette and digested in a 37°C water bath for 20-25 min, mixed gently with a pasteurizing pipette every 5 min. At the end of digestion, added an equal volume of complete medium containing FBS to terminate the digestion. The isolated cells were filtered using 70-μm disposable filter and washed twice with PBS, centrifuged for 5 min at 500 g at 4°C to collect the cell precipitate. To perform erythrocyte lysis, added 1X erythrocyte lysis solution and incubated at room temperature for 5-10min, centrifuged for 5min at 500g, collected cell precipitate. Added PBS (0.04% BSA) and washed 1-2 times, centrifuged for 5 min, discarded the supernatant and resuspend in PBS. For scRNA-seq, the cell density was subsequently changed to 106/ml. Single cell suspensions of vertebral endplate tissue were prepared and tested (cell viability >80%). They were subjected to single cell library construction according to the 10x Single Cell 3' Reagent Kits manual. Using a microfluidic chip, cells and gel beads with cell labeling sequences were wrapped in a droplet, and the cell-wrapped droplet was collected. In the droplet, the cells were lysed so that the mRNA in the cells was connected to the cell Barcode of the bead, forming Single Cell GEMs. These were subjected to RT reactions in the droplet, followed by the disruption of emulsions and cDNA library construction. Cells and reaction reagents were placed in one lane on the microfluidic chip and gel beads in the other lane to form a GEM. Reverse transcription was performed independently in each GEM. Afterward, the labeled cDNA was mixed, then amplified, and the library was constructed. Agilent bioanalyzer and qPCR were used for library quality inspection and library quantification, respectively. The qualified library was sequenced using Novaseq 6000 PE150 mode, and each library measured 60G data.