NextSeq 500 sequencing; ChIP-seq of SATB1-bound regions in human ESC-derived dopaminergic (DA) neurons at different time points. ATAC-Seq and RNA-Seq of DA and cortical neurons (WT and SATB1-KO)
ChIP-seq of SATB1-bound regions in human ESC-derived dopaminergic (DA) neurons at different time points. ATAC-Seq and RNA-Seq of DA and cortical neurons (WT and SATB1-KO)
The human stem cell clone H9 was grown under previously published conditions. In brief, the hESCs wild-type H9 (WA-09), SATB1KO, NURR1-GFP reporter line were maintained using E8-essential medium (Fisher Scientific) without feeder on VTA-N (Fisher Scientific) and passaged every 4-5 days by EDTA. The knockout of the SATB1 gene was performed by using a directed CRISPR/Cas9 approach in the human stem cell line H9. Stem cell clones were screened by Sanger sequencing to validate SATB1-KO. H9 clones were then differentiated into dopaminergic neurons or cortical neurons (predominantly layer V). Midbrain dopamine (mDA) differentiation from hESC was done with an optimized protocol from previously published by our group (Chambers et al., 2009; Kriks et al., 2011). Cortical differentiation from hESC was done with an optimized protocol from previously published by our group (Maroof et al., 2013). References: Chambers, S.M., Fasano, C.A., Papapetrou, E.P., Tomishima, M., Sadelain, M., and Studer, L. (2009). Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling. Nat Biotechnol 27, 275-280. Kriks, S., Shim, J.W., Piao, J., Ganat, Y.M., Wakeman, D.R., Xie, Z., Carrillo-Reid, L., Auyeung, G., Antonacci, C., Buch, A., et al. (2011). Dopamine neurons derived from human ES cells efficiently engraft in animal models of Parkinson's disease. Nature 480, 547-551. Maroof, A.M., Keros, S., Tyson, J.A., Ying, S.W., Ganat, Y.M., Merkle, F.T., Liu, B., Goulburn, A., Stanley, E.G., Elefanty, A.G., et al. (2013). Directed differentiation and functional maturation of cortical interneurons from human embryonic stem cells. Cell Stem Cell 12, 559-572. RNA was extracted using the RNeasy micro plus kit (Qiagen) according to the producers protocol. The DNA library construction protocol for RNA-Seq was performed using the Illumina TrueSeq RNA sequencing kit.
Experimental Factor: immunoprecipitate
not applicable
Experimental Factor: genotype
NURR1::GFP; SATB1 knockout
Experimental Factor: cell type
dopaminergic neuron
Experimental Factor: sampling time point
50
Experimental Factor: protocol
RNA-Seq