GSM9046268: MJe8; Homo sapiens; RNA-Seq
The collected cells were incubated with TrypLE Select in a 37 °C incubator for about 2 minutes, until detachment of the edge of the cells was observed. TrypLE was then removed, and the cells were gently resuspended in DMEM/F12 supplemented with 10% fetal bovine serum (Gibco, 26140079) using a P1000 pipette until no visible aggregates remained. The cell suspension was filtered through a 100 μm cell strainer to eliminate any chunks of cell aggregates. Then, cells were resuspended in 1X phosphate-buffered saline (PBS; Gibco, 14190144) containing 0.04% bovine serum albumin (BSA; SIGMA, A8412). Cell viability was assessed manually using a hemocytometer and confirmed with a BioRad TC20 automated cell counter. The final cell concentration was 1,000 cells/μl, with cell viability exceeding 80%. Single-cell 3′ RNA-seq was performed using the Chromium single-cell system (10X Genomics) and the NexSeq 2000 (Illumina). Dissociated cells were added to a single-cell master mix targeting 10,000 cells, following the Chromium Single Cell 3' v3 protocol (10X Genomics, G000183, Rev C). Along with the single-cell gel beads and oil partitioned in separate wells of a Single Cell B Chip, the single-cell reaction mixture was loaded to the Chromium Controller for Gel Bead-in-Emulsion generation and barcoding. This was followed by cDNA synthesis and library preparation. At each step, cDNA and library quality were examined by Tape station 4200. The final sequencing library was processed using a custom sequencing program on an Illumina NextSeq 2000, with 28-bp plus 91-bp paired-end sequencing, achieving a reading depth of over 30,000 reads per cell.