GSM7346368: 13cRA 5µM+Doxazosin 10µM; Homo sapiens; RNA-Seq
All on-chip liquid handling was performed using the SMARTerTM ICELL8® Single-Cell System, and full-length cDNA synthesis, Illumina P5/P7 index addition, and tagmentation were performed on-chip. Following round 1 PCR, the amplicons were pooled by centrifugation of the chip and purified using Ampure beads. These steps were followed by round 2 PCR amplification off-chip. Library was prepared according to the manufacter's instructions (SMARTerTM ICELL8® Single-Cell Full-Lenght Analysis, Takara). The four single-cell suspensions (CHP134 cells and three treatments) were fluorescently labeled with live/dead stain using READYPROBES VIABILITY BLU/RED1 kit (Invitrogen) and diluted (in 1 ml) to have 1.4 cells in 50 nl. Each cell type suspension was dispensed using the SMARTer ICELL8® Single-Cell System from a 384-well source plate into individually addressable wells in a 5,184 nano-well, 250 nl volume ICELL8 chip (SMARTerTM ICELL8® 250 v Chip). Wells containing individual live cells were identified by imaging using the CellSelect software to generate a well-selection map (filter file:1287 cells), which was used to enable individual addressing of the selected wells for the addition of library preparation reagents. This procedure certifies that only single live cells will be used in the protocol. Library was prepared according to the manufacter's instructions (SMARTerTM ICELL8® Single-Cell Full-Lenght Analysis, Takara). The four single-cell suspensions (CHP134 cells and three treatments) were fluorescently labeled with live/dead stain using READYPROBES VIABILITY BLU/RED1 kit (Invitrogen) and diluted (in 1 ml) to have 1.4 cells in 50 nl. Each cell type suspension was dispensed using the SMARTer ICELL8® Single-Cell System from a 384-well source plate into individually addressable wells in a 5,184 nano-well, 250 nl volume ICELL8 chip (SMARTerTM ICELL8® 250 v Chip). Wells containing individual live cells were identified by imaging using the CellSelect software to generate a well-selection map (filter file:1287 cells), which was used to enable individual addressing of the selected wells for the addition of library preparation reagents. This procedure certifies that only single live cells will be used in the protocol. All on-chip liquid handling was performed using the SMARTerTM ICELL8® Single-Cell System, and full-length cDNA synthesis, Illumina P5/P7 index addition, and tagmentation were performed on-chip. Following round 1 PCR, the amplicons were pooled by centrifugation of the chip and purified using Ampure beads. These steps were followed by round 2 PCR amplification off-chip.