GSM5513589: ICC_plasma; Homo sapiens; RNA-Seq
Fresh tissue samples were collected and immediately stored in the GEXSCOPE Tissue Preservation Solution (Singleron Biotechnologies) at 2-8°C. Prior to tissue dissociation, the specimens were washed with Hanks Balanced Salt Solution (HBSS) for three times and minced into 1–2 mm pieces. The tissue pieces were digested in 2ml GEXSCOPE Tissue Dissociation Solution (Singleron Biotechnologies) at 37°C for 15min in a 15ml centrifuge tube with continuous agitation. Following digestion, a 40-micron sterile strainer (Corning) was used to separate cells from cell debris and other impurities. The cells were centrifuged at 1000 rpm, 4°C , for 5 minutes and cell pellets were resuspended into 1ml PBS (HyClone). To remove red blood cells, 2 mL GEXSCOPE Red Blood Cell Lysis Buffer (Singleron Biotechnologies) was added to the cell suspension and incubated at 25°C for 10 minutes. The mixture was then centrifuged at 1000 rpm for 5 min and the cell pellet resuspended in PBS. Cells were counted with TC20 automated cell counter (Bio-Rad). The concentration of single-cell suspension was adjusted to 1×105 cells/mL in PBS. Single cell suspension was then loaded onto a microfluidic chip and single cell RNA-seq libraries were constructed according to the manufacturer's instructions (Singleron GEXSCOPE Single Cell RNAseq Library Kit, Singleron Biotechnologies). The resulting single cell RNA-seq libraries were sequenced on an Illumina HiSeq X10 instrument with 150bp paired end reads.