Illumina HiSeq 2500 sequencing; KDM5C safeguards the appropriate timing of early neurodevelopment
KDM5C safeguards the appropriate timing of early neurodevelopment
Total RNA was isolated using the TRIZOL reagent according to the manufacturer's instructions (Life Technologies cat no: 15596018). Human iPSC were cultures on irradiated mouse embryonic fibroblast (MEF) feeder cells CF-1 (GlobalStem cat no:GSC-6201G) in DMEM/F12-15mM Hepes (STEMCELL Technologies cat no: 36254) supplemented with 20% KnockOut Serum Replacement (KSR) (Invitrogen cat no:10828-028), 100 mM 2-mercaptoethanol (2ME) (Gibco Cat no: 21985-023), 2mM L-glutamine (corning cat no:25-005-CI), 1XMEM non-essential amino acids (Invitrogen Cat.no:11140050) and 10 ng/ml bFGF (Bio Pioneer Inc cat no: HRP-0011-1). Cells were fed, inspected daily and differentiated cells at the edges of colonies were mechanically removed. Cells were passaged either manually or with collagenase (Invitrogen cat no: 17104-019). The neuronal differentiation was performed according to: Yichen Shi et al., under the title \"Directed differentiation of human pluripotent stem cells to cerebral cortex neurons and neural networks\" published in Nature protocols, 2012 To generate footprint-free genotypically-matched controls, we corrected the KDM5C mutation to the WT sequence using CRISPR/Cas9 and PiggyBac technologies (Yusa, K. Seamless genome editing in human pluripotent stem cells using custom endonuclease-based gene targeting and the piggyBac transposon. Nat Protoc 8, 2061–2078 (2013). The neuronal differentiation was performed according to: Yichen Shi et al., under the title \"Directed differentiation of human pluripotent stem cells to cerebral cortex neurons and neural networks\" published in Nature protocols, 2012 Nucleic acids were exctracted according to NEBNext® Ultra™ Directional RNA Library Prep Kit for Illumina® according to manufacture guidelines. (New England BioLabs cat no: E7420L) Library preparation was performed using the NEBNext® Ultra™ Directional RNA Library Prep Kit for Illumina® according to manufacture guidelines. (New England BioLabs cat no: E7420L and E7490L, E7335L).