GSM7388562: B13F4; Homo sapiens; RNA-Seq
The qualified cells are washed and re-suspended to prepare suitable cell concentration at 700~1200 cells/ml for computer operation of 10X Genomics Chromium™ system. 10X genomics Single Cell Sequencing Using 10X Genomics Chromium™ system, gel Bead with sequence tags, sorted T cells and reagent premixed liquid and oil were loaded into their respective injection channels, a “double cross” cross system was formed through the microfluidic channel network to finally emerge a single cell micro-reaction system GEMs wrapped by oil droplets. The single cell was isolated by gel beads, and then the mRNA molecule was released by cell cleavage and reverse-transcribed into cDNA by the polyT primer. The V (D) J region was amplified by a pair of primers near the 5-terminal UTR region of the V (D) J region and the gene C region. During the amplification process, the unique molecular tag (UMI) of the cell marker and transcript marker were introduced, and the amplified products were digested with restriction endonuclease to produce the second generation sequencing library. Finally, the high-throughput sequencing is carried out by using the double-ended sequencing mode of Illumina platform.