GSM7897845: HET2_D21; Homo sapiens; RNA-Seq
We prepared single cell preparations from fresh cultures by incubation with Accutase 10 min at 37C followed by gently pipetting the cells 3-4 times and an extra 2 min incubation at 37C. After dissociation and pelleting, cells were pipetted to single cells in 500 ul PBS and incubated for staining with Hoechst and PI for 20 min at 37C (ReadyProbes™ Cell Viability Imaging Kit, Blue/Red, R37610 Invitrogen). After washing, we re-suspended the cells in 1ml PBS and filtered through a nylon membrane to remove cell clumps. We used Countess II Automated cell counter (Invitrogen) to quantify cell numbers and cells were dispensed at a concentration of 30.000 cells/ml into ICELL8 Single-Cell System (Takara). The cDNA libraries were generated using the SMART-Seq ICELL8 cx Application Kit protocol provided by the manufacturer