Illumina HiSeq 2500 paired end sequencing; GSM2455567: D26Dn6A01; Homo sapiens; RNA-Seq

D26Dn6A01

Diverse cell types are produced from dorsal and ventral regions of the developing neural tube. In this study we describe a system for generating human inhibitory interneurons by ventralizing human embryonic stem cells in vitro and characterizing the gene expression of the cell types produced over time. We engineered a DCX-Citrine/Y hESC line to sort and characterize progenitor and neuron transcriptomics separately at both the subpopulation and single cell level. The cells generated in vitro were compared to similar populations present in human fetal brain samples by mapping gene expression data from human fetal cells onto the principal component analysis (PCA) space of in vitro-derived populations. Weighted gene co-expression network analysis (WGCNA) was used to determine the discreet cell types present at D24, D54, D100 and D125 of culture, and describe the gene expression changes that occur in progenitor and neuron populations over time. Immature lateral ganglionic eminence and medial ganglionic eminence cells are present at early timepoints, along with MGE-like and dorsal pallium-like neuronal progenitors. At later timepoints we observe the emergence of SST-expressing interneurons, as well as oligodendrocyte and astrocyte progenitors. We also identified genes that were upregulated in somatostatin-expressing interneurons as they mature. Overall design: The transcriptomes of 1732 ventralized single cells were profiled by SmartSeq2 at different timepoints throughout a 125-day differentiation protocol that converted H1 human embryonic stem cells to a variety of ventrally-derived cell types.

Single cell RNAseq characterization of cell types produced over time in an in vitro model of human inhibitory interneuron differentiation.

Submitted by Gene Expression Omnibus on 11-MAR-2017

To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase  (ThermoFisher)  at  37°C.    Light  trituration  using  a P1000  pipette  was  done  every  5  min  until  nearly  all clumps  had  been  dissociated  (up  to  1  h).    Cell  suspension  was  washed  and  filtered  through  a  40  μm  cell  strainer.  Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI.  Single-cell suspensions were loaded onto  a  FACSAria  II  SORP  (Becton  Dickinson)  and  sorted  directly  into  PCR  strip  tubes  or  plates  held  in  chilled aluminum  blocks.    Doublets  and  dead  cells  were  excluded  based  on  forward  scatter,  side  scatter  and  DAPI fluorescence. Sorting was done using the 130  μm nozzle with the sort mode set to single cell. Accuracy of single-cell  sorts  was  confirmed  by  sorting  DAPI-stained  fixed  cells  onto  a  dry  well  of  a  96-well  plate  and  analyzing  by fluorescence microscopy. SmartSeq2 protocol. After  reverse  transcription  and  template  switching,  we amplified  cDNA  with  KAPA  HotStart  HIFI  2×  ReadyMix  (Kapa  Biosystems)  for  22  cycles  for  RNA  from  single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay   Kit  (Thermo  Fisher)  on  an  Enspire  plate  reader  (PerkinElmer).  We  used  1  ng  of  cDNA  to  generate  RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads

Single cell RNAseq characterization of cell types produced over time in an in vitro model of human inhibitory interneuron differentiation.

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