2025-10-30
ENA first public
ERC000011
ENA-CHECKLIST
HARVARD UNIVERSITY
INSDC center name
public
INSDC status
ERS26987656
SRA accession
E-MTAB-15667:SMG1
Submitter Id
ArrayExpress
broker name
WTC, HES3 (NKX2-1/EGFP), H9
cell line
neural cell
cell type
not collected
collection date
human
common name
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.‬ ‭ ‭
description
21 days in vitro
developmental stage
normal
disease
not collected
geographic location (country and/or sea)
Organoid in 96-well plate
growth condition
not applicable
isolate
Homo sapiens
organism
Homo sapiens
organism
hPSC-derived neural organoid
organism part
Homo sapiens
scientific_name
SMG1
title