GSM8510243: iNeuron, replicate 5, scRNA-seq; Homo sapiens; RNA-Seq
Briefly, on the day of collection, papain (Worthington, cat. no. LK003153) was resuspended in 5 mL 1X HBSS (Thermo, cat. no. 2277417) for a concentration of 20 U/mL. A resuspension solution of DMEM (Thermo, cat. no. 10313039) with 10% FBS was also prepared and both solutions were warmed at 37°C for at least 10 minutes. From the plate of neurons, conditioned media (i.e., the media in which the cells were cultured) was separately collected from lentivirus-treated cells as well as the untreated cells. Additionally, 10 µM Y-27632 dihydrochloride ROCK inhibitor (Tocris, cat. no. 1254) and 2% FBS was added to each tube of conditioned media to ensure cell survival and to prevent aggregation in preparation for FACS. Immediately before use, DNase (Worthington, cat. no. LK003153) at 5 μg/mL and Magnesium Chloride (Thermo, cat. no. AM9530G) at 5 mM were added to the papain solution. The cell culture media was aspirated and washed with DPBS (Thermo, cat. no. 14190144), and the papain dissociation solution was added at 500 uL per well. The plate was then placed in the 37°C incubator for 10 minutes, and gently agitated to ensure neuron sheet dissociation. The papain dissociation solution was then quenched in 5 volumes of DMEM and 10% FBS solution, and the neuron sheet was then gently triturated with a P1000 pipette. The lentivirus-treated and untreated cells were then separately transferred to 15 mL conical tubes and centrifuged at 200g for 10 minutes. The supernatant was then carefully removed, and cells were resuspended by slow vortexing in conditioned media solution containing ROCK inhibitor and 2% FBS in preparation for FACS. Live, RFP-expressing cells were FACS isolated by the UNC Advanced Analytics (AA) Core. After FACS, RFP+ cells were immediately processed by the UNC AA Core using the Chromium 10x Genomics 3' v3.1 Dual Index workflow for single cell RNA-seq library generation. Library preparation was done by UNC AA Core according to manufacturer instructions (Chromium 10x Genomics 3' v3.1 Dual Index Workflow).