title: ch.w2.3
organism: Pan troglodytes
description: Protocols: Cells were cultrued in 6-welll 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. iPSCs or ESCs are plated 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.
attributes.tag: ENA first public
attributes.value: 2020-12-16
attributes.tag: ENA last update
attributes.value: 2020-12-10
attributes.tag: External Id
attributes.value: SAMEA7702309
attributes.tag: INSDC center alias
attributes.value: Max Planck Institute for Evolutionary Anthropology, Leipzig, Germany
attributes.tag: INSDC center name
attributes.value: Max Planck Institute for Evolutionary Anthropology, Leipzig, Germany
attributes.tag: INSDC first public
attributes.value: 2020-12-16T04:08:48Z
attributes.tag: INSDC last update
attributes.value: 2020-12-10T13:18:59Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-9233_2:ch.w2.3
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell line
attributes.value: JoC
attributes.tag: cell type
attributes.value: induced excitatory sensory neuron
attributes.tag: common name
attributes.value: chimpanzee
attributes.tag: disease
attributes.value: not available
attributes.tag: genotype
attributes.value: rtTA/Ngn2-positive
attributes.tag: original progenitor cell type
attributes.value: not available
attributes.tag: progenitor cell type
attributes.value: induced pluripotent stem cell
attributes.tag: sample name
attributes.value: E-MTAB-9233_2:ch.w2.3
attributes.tag: stimulus
attributes.value: neuronal differentiation by Ngn2 induction