Illumina HiSeq 4000 sequencing; Gene expression profiling (RNA-seq) of human iPSCs, iPSC-derived ventralized cells, neurons, and hypothalamic neurons where regions of the genome have been deleted that contain the single nucleotide polymorphisms rs9972768 or rs2650492
Gene expression profiling (RNA-seq) of human iPSCs, iPSC-derived ventralized cells, neurons, and hypothalamic neurons where regions of the genome have been deleted that contain the single nucleotide polymorphisms rs9972768 or rs2650492
Fluorescently tagged crRNA-Atto550 and cas9 protein were purchased from IDT. Guides were designed using IDT software in order to maximize cutting efficacy and minimize off target cleavage using their RNP system. Two guides were designed per region in order to delete the enhancers from their endogenous context. IDT crRNA and tracrRNA were complexed according to manufacturerâ€™s instructions. The day of transfection, 50uM of each guide and cas9 protein were combined and incubated at room temperature for 20 minutes to form RNPs. Each guide was complexed with cas9 in individual reactions. 900k NA19101 human iPSC cells were harvested and nucleofected using a Lonza nucleofector 2b device with the program A23 and the two RNP complexes. These cells were plated into one 22cm2 flask. Once recovered, 40k cells were split into a 100cm2 flask to achieve single cell colonies. Colonies were then picked and transferred into 48 well plates to grow independently. Colonies were screened for the presence of homozygous deletion bands using PCR. Homozygous deletion colonies were grown, transfected, split, and treated identically to WT cells in order to mitigate RNA-seq batch effects for the differentiation. Cells remained frozen until all timepoints were collected.  RNA was extracted using the Qiagen RNeasy Kit and RNA-quality was assessed via an Agilent Bioanalyzer RNA Chip. 1ug of RNA was used for input into RNA-seq. RNA-seq was performed using the NEB Next Ultra II Directional RNA-seq kit