GSM5161290: VCaP DMSO [scRNA-seq]; Homo sapiens; RNA-Seq
Cells were trypsinized with TrypLETM Express Enzyme (ThermoFisher Scientific, #12604021), spun down (5 min at 300xg) and washed with 0.1% BSA-PBS. After pelleting, the cells were resuspended in plain PBS and passed through a 40-micron filter. The number of viable cells was estimated with the use of trypan blue staining and Fuchs-Rosenthal hemocytometer chamber. The concentration of cells was brought down to 3x105 cells/mL in 0.1% BSA-PBS. For single-cell encapsulation, single-cell suspension, beads in lysis buffer and oil were connected with the loops and tubing to the Mitos P pumps and run through the glass microfluidic chip at the following flow rates: 100μL/min (Oil channel), 20μL/min (Bead channel); 350 mbar (Cell channel). Droplets were separated by centrifugation and beads counted with the use of Fuchs-Rosenthal hemocytometer chamber and up to 90000 beads were collected into one tube for Reverse Transcription reaction, exonuclease treatment, and amplification of cDNA library according to the original protocol (Macosko et al., 2015). Tagmentation of cDNA was performed with the Nextera XT DNA Library Preparation Kit (Illumina, #FC-131-1024). The PCR product was cleaned-up with AMPure XP beads and eluted in 10μL H2O. Samples were prepared with Drop-seq (Macosko et al., 2015) using the Dolomite cell encapsulation system (Dolomite Bio).