GSM5988612: kidney, s200813M4; Homo sapiens; RNA-Seq
Single-cell suspensions were obtained by being placed in a 15ml centrifuge tube with a maintained constant agitation at 37°C for 15min and digested in 2ml GEXSCOPE Tissue Dissociation Solution (Singleron Biotechnologies). Then samples were filtered via a 40-mm sterile cell strainer (Corning) which removed cell debris and additional impurities, before being centrifuged at 1000 rpm (300 x g for 5 minutes) at 4°C. In addition, the cell pellets were re-suspended with the use of a 1ml phosphate buffer saline (PBS HyClone). Next came cell suspension incubation via using a 2ml GEXSCOPE Red Blood Cell Lysis Buffer (Singleron Biotechnologies) at 25°C for a total of 10 minutes to extract and discard the red blood cells. After which, the cells were centrifuged at 1000 rpm (300 x g for 5 min) and re-suspended in cold PBS for downstream analyses. Ultimately, quantification of cell yields was subsequently conducted by using the TC20 automated cell counter (Bio-Rad), and live cells were determined by trypan blue staining (Gibco). Once cell viability exceeded 85%, sample processing was conducted. RNA-seq libraries were prepared by drawing upon the Singleron GEXSCOPE Single Cell RNA-seq Library Kit (Singleron Biotechnologies) which is comprised of   mRNA (UMI and mRNA trapping, cell lysis, reverse transcription mRNA into cDNA and amplification, labeling cells (barcode) and fragment cDNA. Individual libraries were diluted to 4 nM and pooled for sequencing. Pools were sequenced on an Illumina HiSeq X with 150 bp paired end reads.