E-MTAB-6018:Chap_D4.fastq.gz_s
Illumina HiSeq 2500 sequencing; Time line RNAseq of iPSC-derived neurons from ASD and control subjects.
Time line RNAseq of iPSC-derived neurons from ASD and control subjects.
Chap_D4.fastq.gz_s
We first used fluorescence-activated cell sorting (FACS) to purify a homogeneous population of NSCs (D0) based on the expression of the cell-surface markers CD184+/CD271-/CD44-/CD24-/CD15+. To trace ASD and control neurons over time, we performed a series of retroviral lineage-tracing experiments to trace the progenies of dividing NSCs using a retroviral vector expressing a membrane-tagged enhanced green fluorescent protein (eGFP) (CAG::LckN-eGFP). As differentiating neurons express PSA-NCAM on the cell surface, we established a FACS-based protocol for purification of defined subpopulations of retrovirally labeled eGFP+/PSA-NCAM+ double-positive neurons after 2, 4, 7 and 14 (D2, 4, 7 and 14) days of differentiation. IPSCs were sorted based on the expression of SSEA-4 and TRA1-81 and maturing iPSC-iNs were collected at the indicated days after induction by sorting for eGFP (indicative for the Ngn2 transgene)- and PSA-NCAM-positive cells. NSC-derived neurons: We differentiated iPSCs into NSCs by following a default differentiation protocol. Briefly, iPSC colonies were detached by collagenase (Gibco) to form embryoid bodies (EBs) and then cultured in suspension in the presence of 1 μM dorsomorphin (Cayman Chemical). After floating for seven days, EBs were attached on poly-ornithine (10 μg/ml, Life Technologies) and laminin (5 μg/ml, Life Technologies) coated surfaces. At day 16, emerging rosette colonies were manually detached, gently dissociated with accutase (Innovative Cell Technologies) and plated as NSC monolayers onto coated dishes for further neuronal differentiation. We first used fluorescence-activated cell sorting (FACS) to purify a homogeneous population of NSCs based on the expression of the cell-surface markers CD184+/CD271-/CD44-/CD24-/CD15+. To trace ASD and control neurons over time, we performed a series of retroviral lineage-tracing experiments to trace the progenies of dividing NSCs using a retroviral vector expressing a membrane-tagged enhanced green fluorescent protein (eGFP) (CAG::LckN-eGFP). Briefly, the sequence of the membrane localization domain from Lck (LckN) was linked to the eGFP sequence and cloned into the Moloney Murine Leukemia Virus (MMLV)-based retroviral plasmid resulting in pRV-CAG:LckN-eGFP (CAG:LckN-eGFP). As differentiating neurons express PSA-NCAM on the cell surface, we established a FACS-based protocol for purification of defined subpopulations of retrovirally labeled eGFP+/PSA-NCAM+ double-positive neurons after 2, 4, 7 and 14 days of differentiation. NSCs and maturing neuron cultures as well as iPSCs and iPSC-iNs were detached using accutase (Innovative Cell Technologies) and then washed and stained with fluorophore-coupled antibodies (Extended Data Table 11) for 45 min at 4ºC in phosphate buffered saline (PBS). Cells were washed, suspended in PBS and filtered using a 40-μm cell strainer. The desired cell populations were sorted directly into Trizol-LS (Life Technologies). RNA was isolated according to the manufacturer's instructions and subsequently digested with TURBO DNase to remove trace quantities of DNA (ambion, Life Technologies). Before RNA-seq library preparation, assessment of RNA integrity numbers (RIN) was performed using the Agilent TapeStation. Libraries were prepared using the TruSeq Stranded mRNA Sample Prep Kit according to the manufacturer's instructions (Illumina).
Laboratory of Genetics - Gage The Salk Institute for Biological Studies
E-MTAB-6018:Chap_D4.fastq.gz_s
Experimental Factor: disease
normal
Experimental Factor: progenitor cell type
neuronal stem cell
Experimental Factor: cell type
neuron
Experimental Factor: time
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