OG2
Homo sapiens
Protocols: Organoids were harvested after 21 days in culture and pooled prior to dissociation into single cells. hPSCs were aggregated into embryoid bodies in iPS Brew medium, then exposed to Neural Induction Medium for 14 days and finally changed into Neural Differentiation Medium with Vitamin A until day 21.  Morphogen treatments were applied from day 0 to day 20 of culture. Each condition featured a systematic variation in morphogen timing, concentration or combination. Cells were lysed immediately after GEM generation as indicated in the 10X Genomics Chromium Next GEM Single Cell 3' v3.1 kit protocol. Once‬‭ cells‬‭ were‬‭ encapsulated‬‭ in‬‭ GEMs,‬‭ an‬‭ RT‬‭ step‬‭ was‬‭ run‬‭ to‬‭ convert‬‭ polyadenylated‬‭ mRNAs‬‭ into‬‭ full-length‬‭ cDNA‬‭ sequences‬‭ with‬‭ a‬‭ UMI‬‭ and‬‭ cellular‬‭ barcode.‬‭ cDNA‬‭ products‬‭ were‬‭ then‬‭ purified‬‭ with‬‭ magnetic‬‭ beads‬‭ (Dynabeads‬‭ MyOne‬‭ SILANE,‬‭ manufactured‬‭ by‬‭ Thermo‬‭ Fisher‬‭ but‬‭ included‬‭ in‬‭ 10X‬‭ Genomics‬‭ kits)‬‭ and‬‭ further‬‭ amplified‬‭ in‬‭ an‬‭ extra‬‭ PCR‬‭ step‬‭ using‬‭ a‬‭ partial‬‭ TruSeq‬‭ read‬‭ 1‬‭ and‬ partial‬‭ TSO‬‭ (Template-Switch‬‭ Oligo)‬‭ as‬‭ primers.‬‭ Another‬‭ purification‬‭ step‬‭ based‬‭ on‬‭ construct‬ size‬‭ was‬‭ performed‬‭ with‬‭ SPRIselect‬‭ reagent‬‭ (Beckman‬‭ Coulter)‬‭ prior‬‭ to‬‭ quantification‬‭ and‭ quality control with a Bioanalyzer High Sensitivity chip (2100 Bioanalyzer, Agilent).‬ One-quarter‬‭ of‬‭ the‬‭ cDNA‬‭ from‬‭ each‬‭ 10X‬‭ sample‬‭ was‬‭ carried‬‭ over‬‭ for‬‭ gene‬‭ expression‬‭ library‬‭ construction.‬‭ In‬‭ short,‬‭ cDNA‬‭ was‬‭ fragmented‬‭ to‬‭ eliminate‬‭ the‬‭ TSO‬‭ incorporated‬‭ in‬‭ the‬ previous‬‭ step‬‭ and‬‭ ends‬‭ were‬‭ repaired‬‭ and‬‭ A-tailed.‬‭ Reaction‬‭ products‬‭ were‬‭ purified‬‭ with‬ SPRIselect‬‭ reagent‬‭ and‬‭ Truseq‬‭ read‬‭ 2‬‭ adaptors‬‭ were‬‭ ligated,‬‭ one‬‭ of‬‭ them‬‭ being‬‭ a‬‭ partial‬ read‬‭ to‬‭ provide‬‭ an‬‭ overhang‬‭ for‬‭ the‬‭ following‬‭ PCR.‬‭ Another‬‭ cleanup‬‭ step‬‭ with‬‭ SPRIselect‬‭ was‬‭ performed‬‭ before‬‭ the‬‭ Sample‬‭ Index‬‭ PCR‬‭ when‬‭ P5‬‭ and‬‭ P7‬‭ sequences‬‭ (for‬‭ Illumina‬‭ sequencing)‬‭ and‬‭ sample‬‭ indices‬‭ (to‬‭ allow‬‭ for‬‭ multiplexing‬‭ in‬‭ the‬‭ sequencer)‬‭ were‬‭ included‬‭ in‬‭ the‬‭ construct.‬‭ The‬‭ final‬‭ product‬‭ was‬‭ purified‬‭ with‬‭ SPRIselect‬‭ magnetic‬‭ beads‬‭ and‬‭ a‬‭ concentration‬‭ and‬‭ quality‬‭ check‬‭ were‬‭ performed‬‭ by‬‭ running‬‭ a‬‭ Bioanalyzer‬‭ High‬‭ Sensitivity‬‭ chip before sequencing.‬ ‭ ‭
ENA first public
2025-10-30
INSDC center name
HARVARD UNIVERSITY
INSDC status
public
Submitter Id
E-MTAB-15667:OG2
broker name
ArrayExpress
cell line
WTC, HES3 (NKX2-1/EGFP)
cell type
neural cell
collection date
not collected
common name
human
developmental stage
21 days in vitro
disease
normal
geographic location (country and/or sea)
not collected
growth condition
Organoid in 96-well plate
isolate
not applicable
organism part
hPSC-derived neural organoid
sample name
E-MTAB-15667:OG2
scientific_name
Homo sapiens