Homo sapiens
Protocols: 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).
ENA-FIRST-PUBLIC
2023-10-13T20:40:13Z
ENA-LAST-UPDATE
2023-10-13T20:40:13Z
External Id
SAMEA113611964
INSDC center alias
Division of Newborn Medicine and Epigenetics Program, Boston Children's Hospital, Boston, MA 02115, USA; Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA.
INSDC center name
Division of Newborn Medicine and Epigenetics Program, Boston Children's Hospital, Boston, MA 02115, USA; Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA.
INSDC first public
2023-10-13T20:40:13Z
INSDC last update
2023-10-13T20:40:13Z
INSDC status
public
Submitter Id
E-MTAB-7551:set1-Mutant_day60
broker name
ArrayExpress
cell_type
fibroblast
collection date
not collected
common name
human
disease
intellectual disability
genotype
KDM5C c.2T>C
geographic location (country and/or sea)
not collected
individual
donor 1
isolate
not applicable
organism part
skin
sample name
E-MTAB-7551:set1-Mutant_day60
scientific_name
Homo sapiens
sex
male