GSM6647951: LNCaP cells, DMSO treatment; Homo sapiens; RNA-Seq
The 15 cm cell culture dishes were removed from the 37 °C incubator, medium was removed via aspiration, and cells were immediately washed once with 10 mL of 37 °C phosphate buffered saline (PBS), pH 7.4. PBS was removed via aspiration and 3 mL 0.25% trypsin (Corning) was added to the dishes. Dishes were rotated for complete coverage of the trypsin and incubated on the benchtop for 30 seconds. Roughly 2 mL of the trypsin was removed via aspiration, and dishes were transferred to a 37 °C incubator for exactly 4 minutes. 10 mL of 4 °C RPMI supplemented with 10% FBS was used for resuspension of the trypsinized cell suspension. Viability was checked on Countess II FL automated cell counter to ensure >90% viability. Cells were then centrifuged 4 minutes at 400x g at 4 °C to pellet the cells. Supernatant was aspirated and discarded, then cells were washed  with 1 mL 4 °C complete RPMI three times. Final resuspension of cells in 400 μL 4 °C complete RPMI was performed, and cell homogeneity and viability were checked to ensure singlet cells at >90% viability would be delivered to Indiana University School of Medicine Chemical Genomics Facility for 10X library preparation. The single cell gene expression analysis was conducted using a 10X Chromium single cell system (10X Genomics, Inc) and a NovaSeq 6000 sequencer (Illumina, Inc). To maximize quality of cell suspension, cell harvest was optimized to retain >95% viability at time of harvest following three washes in RPMI/10%FBS. Following storage on ice for delivery of cells to 10X sequencing facility, the single cell suspension was subsequently washed a fourth time with RPMI/10% FBS, recentrifuged, and cell debris, dead cells and cell aggregates were removed by discarding supernatant and resuspending cells. Each clean single cell suspension was then be counted with hemocytometer under microscope for cell number and cell viability. The single cell suspension viability was over >90% and minimal cell debris and aggregation were ensured prior to loading. A target of 10,000 cells was used for loading; in practice, following final analysis, between 6,890 and 11,620 individual cells were identified per sample after all processing steps were completed. The cells were applied to a single cell master mix with lysis buffer and reverse transcription reagents, following the Chromium NextGEM Single Cell 3' Reagent Kits User Guide, CG000204 Rev D (10X Genomics, Inc). Along with the single cell gel beads and partitioning oil, the single cell master mixture containing the single cell suspension was dispensed onto a Single Cell Chip G in separate wells, and the chip loaded to the Chromium Controller for GEM generation and barcoding, followed by cDNA synthesis and library preparation. At each step, the quality of cDNA and library was be examined by Bioanalyzer and Qubit. The resulting library was sequenced in a custom program for 28b plus 91b paired-end sequencing on Illumina NovaSeq 6000. Depending on targeted cell recovery, roughly 33,000 – 82,000 reads per cell were generated and 91% of the sequencing reads reached Q30 (99.9% base call accuracy). A Phred quality score (Q score) was used to measure the quality of sequencing.