Homo sapiens
Protocols: The hIPSC cell line used has been published in the following paper: NGN2 OPTi-OX hiPSCs https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5390118/ . WT i-neurons in control (37 ºC throughout, collect at day 18), cooled (72 h at 32 ºC, day 15-18, collect at day 18) and rewarmed (72 h at 32 ºC, day 15-18, followed by 72 h at 37 ºC, day 18-21, collect at day 21) conditions were collected by detaching from tissue culture dishes. hiPSCs were enzymatically detached and dissociated into single cells using Accutase and plated into GelTrex (1:100 dilution)-coated culture plates in TeSR-E8 medium supplemented with 10 µM Rho-associated protein kinase (ROCK) inhibitor. After 24 hours (day 1), TeSR-E8 medium was changed to DMEM/F12 medium with GlutaMAX, supplemented with 1x N-2 supplement, 1x Non-Essential Amino Acids, 50 nM 2-Mercaptoethanol, 100 U/mL Penicillin-Streptomycin, and 1 µg/mL Doxycycline Hyclate (Dox) (iN-1 medium). After 24 hours (day 2), the medium was replaced with the same medium as the previous day. From day 3 to day 6, the culture was fed daily with Neurobasal medium supplemented with 1x B-27 supplement (minus vitamin A), 1x GlutaMAX, 50 nM 2-Mercaptoethanol, 100 U/mL Penicillin-Streptomycin, and 1 µg/mL Dox, 10 ng/mL Neurotrophin-3 (NT-3), and 10 ng/mL Brain-derived neurotrophic factor (BDNF) (iN-2 medium). After day 6, the medium was changed every other day. Total RNA was extracted with RNeasy Plus Mini Kit (Qiagen) following the manufacturer's instructions. RNA concentrations of individual samples were measured using Qubit RNA Broad Range Assay Kit and their integrity was determined using Agilent TapeStation System. The library was prepared using Illumina TruSeq Stranded mRNA Library Prep following the manufacturer's instructions.
ENA-FIRST-PUBLIC
2023-05-04T12:01:11Z
ENA-LAST-UPDATE
2023-05-04T12:01:11Z
External Id
SAMEA112194107
INSDC center name
The Francis Crick Institute UK Dementia Research Institute at King's College London
INSDC first public
2023-05-04T12:01:11Z
INSDC last update
2023-05-04T12:01:11Z
INSDC status
public
Submitter Id
E-MTAB-12402:WT_RW_2
broker name
ArrayExpress
cell_line
NGN2 OPTi-OX hiPSC
cell_type
cortical neuron
common name
human
developmental stage
adult
disease
normal
genotype
wild type genotype
progenitor cell type
induced pluripotent stem cell
sample name
E-MTAB-12402:WT_RW_2
scientific_name
Homo sapiens
sex
male