Homo sapiens
Protocols: Generation of human somitoids without Matrigel. Once human iPSCs reached 50-70% confluence, the cells were washed with PBS(-) twice and incubated with 2 ml 0.5 mM EDTA in PBS at 37ºC for 6-7 min. The cells were mechanically dissociated by pipetting and transferred into 8 ml of the N2B27 medium described below. The cell suspension was centrifuged at 152 xg for 3 min and resuspended into 10 ml of N2B27 medium. The washing step was repeated once, and the supernatant was completely removed. The cell pellet was resuspended into 150-500 μl of the somitoid induction medium described below with 10 µM Y27632. The cell concentration was calculated and adjusted to 7 cells/ml with an adequate amount of pre-warmed induction medium with Y27632. Then, 50 µl of the cell suspension (= 350 cells) was aliquoted into each well of a U-Shaped-Bottom, 96-well-plate (Nunclon Sphera 96U-well plate, Thermo Scientific, 174925) by using a multichannel pipette. To settle down the cells on the bottom of the plate, the 96-well-plate was centrifuged at 152 xg for 2 min before incubation. One day after aggregate formation, 150 µl of fresh somitoid induction medium without Y27632 was added to each well. From day 2, 150 µl of the medium was carefully removed and replaced with 150 µl of fresh N2B27 medium without disturbing the aggregate. On day7, somitoids were collected. RNA extraction included in the single-cell nucleic acid library construction protocol cDNA libraries were constructed with Chromium Single Cell 3' GEM, Library & Gel Bead Kit v3 (10x Genomics) according to manufacturer's protocol.
ENA first public
2022-04-01
ENA last update
2022-04-01
External Id
SAMEA12131273
INSDC center alias
EMBL
INSDC center name
European Molecular Biology Laboratory
INSDC first public
2022-04-01T00:17:00Z
INSDC last update
2022-04-01T00:17:00Z
INSDC status
public
Submitter Id
E-MTAB-11334:Somitoids_S02
broker name
ArrayExpress
cell line
Human iPS cells (201B7 with HES7 promoter-luciferase reporter and the fluorescent nuclear reporter)
cell type
induced pluripotent stem cell
common name
human
disease
normal
growth condition
Without-Matrigel; +CHIR99021_8-10
sample name
E-MTAB-11334:Somitoids_S02