Illumina HiSeq 2500 paired end sequencing; Single-cell RNA sequencing of NGN2-induced neurons from human and chimpanzee pluripotent stem cells
Single-cell RNA sequencing of NGN2-induced neurons from human and chimpanzee pluripotent stem cells
Cells were cultured in 6-well plates and dissociated into single cells to perform a 10x analysis. All reagents were filtered through a 0.22 µm filter. Each well was washed with DPBS before the cells were incubated for 5 min in 1 ml EDTA 1 % accutase/well. Using 1 ml pipette tip, cells were carefully detached from the plate by continuous rinsing to obtain a cell suspension enriched for iNs. All wells of one cell line were pooled and centrifuged for 5 min at 300 g. Then, the supernatant was discarded and the pellet was resuspended in 1 ml accutase with 0.3 µl DNAse (2000 U/µl) for 5 min. The cell pellet was washed 2 3 times in 5 ml Neurobasal medium supplemented with 10% FCS followed by centrifugation for 5 min at 300 g. Then, the pellet was pipetted 20 times until complete resuspension in 20-00 µl Neurobasal medium supplemented with 10% FCS and filtered through a 30 µm filter. Finally, we centrifuged cells on a Percoll gradient for 5 min at 300g to enrich for healthy cells and remove debris. The Percoll gradient consisted of three layers. Lowe layer: 4 ml wash medium and 1 ml Percoll solution; middle layer: 4 ml wash medium and 0.75 ml Percoll solution; upper layer: 4 ml wash medium and 0.5 ml Percoll solution. Cells were analyzed using Trypan Blue assay and counted with the automated cell counter Countess (Thermo Fisher) and diluted to yield a concentration of 500 to 1000 cells/ml to obtain approximately 3000 cells per lane of a 10X microfluidic chip device.  We plated iPSCs or ESCs as single cells and initiated the neuronal differentiation by doxycycline-induction of NGN2. After two days of culturing we added primary cortical rat astrocytes to the system. One day after astrocyte plating we stopped proliferation of undifferentiated stem cells and proliferating astrocytes by supplementing the growth medium with 2 µM cytosine arabinoside (Ara-C). iNs were kept for two to eight weeks in culture with 50% medium changed every two to three days.  Single cell RNAs were extracted using the 10X Chromium Single Cell 3' v2 Kit following the manufacturer's instructions.  Single cell gene expression libraries were generated using the 10X Chromium Single Cell 3' v2 Kit following the manufacturer's instructions. Quantification and quality control of libraries was performed using a High Sensitivity DNA chip for Agilent's Bioanalyzer.
Experimental Factor: cell line
409B2, SandraA
Experimental Factor: growth condition
neuronal differentiation
Experimental Factor: time
14