ERS1886913 SAMEA104227895 9606 Homo sapiens Protocols: 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). ENA first public 2018-01-01 ENA last update 2017-08-25 External Id SAMEA104227895 INSDC center alias Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC center name Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC first public 2018-01-01T17:01:42Z INSDC last update 2017-08-25T17:29:33Z INSDC status public Submitter Id E-MTAB-6018:Able_D0.fastq.gz broker name ArrayExpress cell type neuronal stem cell common name human disease autism spectrum disorder genotype pRV-CAG:LckN-eGFP individual Able organism part skin phenotype CD184+/CD271-/CD44-/CD24-/CD15+ progenitor cell type induced pluripotent stem cell sample name E-MTAB-6018:Able_D0.fastq.gz time 0 ERS1886914 SAMEA104227896 9606 Homo sapiens Protocols: 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). ENA first public 2018-01-01 ENA last update 2017-08-25 External Id SAMEA104227896 INSDC center alias Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC center name Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC first public 2018-01-01T17:01:42Z INSDC last update 2017-08-25T17:29:33Z INSDC status public Submitter Id E-MTAB-6018:Acai_D0.fastq.gz broker name ArrayExpress cell type neuronal stem cell common name human disease autism spectrum disorder genotype pRV-CAG:LckN-eGFP individual Acai organism part skin phenotype CD184+/CD271-/CD44-/CD24-/CD15+ progenitor cell type induced pluripotent stem cell sample name E-MTAB-6018:Acai_D0.fastq.gz time 0 ERS1886915 SAMEA104227897 9606 Homo sapiens Protocols: 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). ENA first public 2018-01-01 ENA last update 2017-08-25 External Id SAMEA104227897 INSDC center alias Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC center name Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC first public 2018-01-01T17:01:42Z INSDC last update 2017-08-25T17:29:33Z INSDC status public Submitter Id E-MTAB-6018:Aero_D0.fastq.gz broker name ArrayExpress cell type neuronal stem cell common name human disease autism spectrum disorder genotype pRV-CAG:LckN-eGFP individual Aero organism part skin phenotype CD184+/CD271-/CD44-/CD24-/CD15+ progenitor cell type induced pluripotent stem cell sample name E-MTAB-6018:Aero_D0.fastq.gz time 0 ERS1886916 SAMEA104227898 9606 Homo sapiens Protocols: 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). ENA first public 2018-01-01 ENA last update 2017-08-25 External Id SAMEA104227898 INSDC center alias Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC center name Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC first public 2018-01-01T17:01:42Z INSDC last update 2017-08-25T17:29:33Z INSDC status public Submitter Id E-MTAB-6018:Agua_D0.fastq.gz broker name ArrayExpress cell type neuronal stem cell common name human disease autism spectrum disorder genotype pRV-CAG:LckN-eGFP individual Agua organism part skin phenotype CD184+/CD271-/CD44-/CD24-/CD15+ progenitor cell type induced pluripotent stem cell sample name E-MTAB-6018:Agua_D0.fastq.gz time 0 ERS1886917 SAMEA104227899 9606 Homo sapiens Protocols: 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). ENA first public 2018-01-01 ENA last update 2017-08-25 External Id SAMEA104227899 INSDC center alias Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC center name Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC first public 2018-01-01T17:01:42Z INSDC last update 2017-08-25T17:29:33Z INSDC status public Submitter Id E-MTAB-6018:Ahoy_D0.fastq.gz broker name ArrayExpress cell type neuronal stem cell common name human disease autism spectrum disorder genotype pRV-CAG:LckN-eGFP individual Ahoy organism part skin phenotype CD184+/CD271-/CD44-/CD24-/CD15+ progenitor cell type induced pluripotent stem cell sample name E-MTAB-6018:Ahoy_D0.fastq.gz time 0 ERS1886918 SAMEA104227900 9606 Homo sapiens Protocols: 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). ENA first public 2018-01-01 ENA last update 2017-08-25 External Id SAMEA104227900 INSDC center alias Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC center name Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC first public 2018-01-01T17:01:42Z INSDC last update 2017-08-25T17:29:33Z INSDC status public Submitter Id E-MTAB-6018:Apex_D0.fastq.gz broker name ArrayExpress cell type neuronal stem cell common name human disease autism spectrum disorder genotype pRV-CAG:LckN-eGFP individual Apex organism part skin phenotype CD184+/CD271-/CD44-/CD24-/CD15+ progenitor cell type induced pluripotent stem cell sample name E-MTAB-6018:Apex_D0.fastq.gz time 0 ERS1886919 SAMEA104227901 9606 Homo sapiens Protocols: 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). ENA first public 2018-01-01 ENA last update 2017-08-25 External Id SAMEA104227901 INSDC center alias Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC center name Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC first public 2018-01-01T17:01:42Z INSDC last update 2017-08-25T17:29:33Z INSDC status public Submitter Id E-MTAB-6018:Arch_D0.fastq.gz broker name ArrayExpress cell type neuronal stem cell common name human disease autism spectrum disorder genotype pRV-CAG:LckN-eGFP individual Arch organism part skin phenotype CD184+/CD271-/CD44-/CD24-/CD15+ progenitor cell type induced pluripotent stem cell sample name E-MTAB-6018:Arch_D0.fastq.gz time 0 ERS1886920 SAMEA104227902 9606 Homo sapiens Protocols: 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). ENA first public 2018-01-01 ENA last update 2017-08-25 External Id SAMEA104227902 INSDC center alias Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC center name Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC first public 2018-01-01T17:01:42Z INSDC last update 2017-08-25T17:29:33Z INSDC status public Submitter Id E-MTAB-6018:Avid_D0.fastq.gz broker name ArrayExpress cell type neuronal stem cell common name human disease autism spectrum disorder genotype pRV-CAG:LckN-eGFP individual Avid organism part skin phenotype CD184+/CD271-/CD44-/CD24-/CD15+ progenitor cell type induced pluripotent stem cell sample name E-MTAB-6018:Avid_D0.fastq.gz time 0 ERS1886921 SAMEA104227903 9606 Homo sapiens Protocols: 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). ENA first public 2018-01-01 ENA last update 2017-08-25 External Id SAMEA104227903 INSDC center alias Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC center name Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC first public 2018-01-01T17:01:42Z INSDC last update 2017-08-25T17:29:33Z INSDC status public Submitter Id E-MTAB-6018:Cent_D0.fastq.gz broker name ArrayExpress cell type neuronal stem cell common name human disease normal genotype pRV-CAG:LckN-eGFP individual Cent organism part skin phenotype CD184+/CD271-/CD44-/CD24-/CD15+ progenitor cell type induced pluripotent stem cell sample name E-MTAB-6018:Cent_D0.fastq.gz time 0 ERS1886922 SAMEA104227904 9606 Homo sapiens Protocols: 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). ENA first public 2018-01-01 ENA last update 2017-08-25 External Id SAMEA104227904 INSDC center alias Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC center name Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC first public 2018-01-01T17:01:42Z INSDC last update 2017-08-25T17:29:33Z INSDC status public Submitter Id E-MTAB-6018:Chap_D0.fastq.gz broker name ArrayExpress cell type neuronal stem cell common name human disease normal genotype pRV-CAG:LckN-eGFP individual Chap organism part skin phenotype CD184+/CD271-/CD44-/CD24-/CD15+ progenitor cell type induced pluripotent stem cell sample name E-MTAB-6018:Chap_D0.fastq.gz time 0 ERS1886923 SAMEA104227905 9606 Homo sapiens Protocols: 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). ENA first public 2018-01-01 ENA last update 2017-08-25 External Id SAMEA104227905 INSDC center alias Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC center name Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC first public 2018-01-01T17:01:42Z INSDC last update 2017-08-25T17:29:33Z INSDC status public Submitter Id E-MTAB-6018:Clay_D0.fastq.gz broker name ArrayExpress cell type neuronal stem cell common name human disease normal genotype pRV-CAG:LckN-eGFP individual Clay organism part skin phenotype CD184+/CD271-/CD44-/CD24-/CD15+ progenitor cell type induced pluripotent stem cell sample name E-MTAB-6018:Clay_D0.fastq.gz time 0 ERS1886924 SAMEA104227906 9606 Homo sapiens Protocols: 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). ENA first public 2018-01-01 ENA last update 2017-08-25 External Id SAMEA104227906 INSDC center alias Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC center name Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC first public 2018-01-01T17:01:42Z INSDC last update 2017-08-25T17:29:33Z INSDC status public Submitter Id E-MTAB-6018:Clue_D0.fastq.gz broker name ArrayExpress cell type neuronal stem cell common name human disease normal genotype pRV-CAG:LckN-eGFP individual Clue organism part skin phenotype CD184+/CD271-/CD44-/CD24-/CD15+ progenitor cell type induced pluripotent stem cell sample name E-MTAB-6018:Clue_D0.fastq.gz time 0 ERS1886925 SAMEA104227907 9606 Homo sapiens Protocols: 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). ENA first public 2018-01-01 ENA last update 2017-08-25 External Id SAMEA104227907 INSDC center alias Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC center name Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC first public 2018-01-01T17:01:42Z INSDC last update 2017-08-25T17:29:33Z INSDC status public Submitter Id E-MTAB-6018:Cove_D0.fastq.gz broker name ArrayExpress cell type neuronal stem cell common name human disease normal genotype pRV-CAG:LckN-eGFP individual Cove organism part skin phenotype CD184+/CD271-/CD44-/CD24-/CD15+ progenitor cell type induced pluripotent stem cell sample name E-MTAB-6018:Cove_D0.fastq.gz time 0 ERS1886926 SAMEA104227908 9606 Homo sapiens Protocols: 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). ENA first public 2018-01-01 ENA last update 2017-08-25 External Id SAMEA104227908 INSDC center alias Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC center name Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC first public 2018-01-01T17:01:42Z INSDC last update 2017-08-25T17:29:33Z INSDC status public Submitter Id E-MTAB-6018:Able_D14.fastq.gz broker name ArrayExpress cell type neuron common name human disease autism spectrum disorder genotype pRV-CAG:LckN-eGFP individual Able organism part skin phenotype Ngn2+/PSA-NCAM+ progenitor cell type neuronal stem cell sample name E-MTAB-6018:Able_D14.fastq.gz time 14 ERS1886927 SAMEA104227909 9606 Homo sapiens Protocols: 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). ENA first public 2018-01-01 ENA last update 2017-08-25 External Id SAMEA104227909 INSDC center alias Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC center name Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC first public 2018-01-01T17:01:42Z INSDC last update 2017-08-25T17:29:33Z INSDC status public Submitter Id E-MTAB-6018:Able_D2.fastq.gz broker name ArrayExpress cell type neuron common name human disease autism spectrum disorder genotype pRV-CAG:LckN-eGFP individual Able organism part skin phenotype Ngn2+/PSA-NCAM+ progenitor cell type neuronal stem cell sample name E-MTAB-6018:Able_D2.fastq.gz time 2 ERS1886928 SAMEA104227910 9606 Homo sapiens Protocols: 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). ENA first public 2018-01-01 ENA last update 2017-08-25 External Id SAMEA104227910 INSDC center alias Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC center name Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC first public 2018-01-01T17:01:42Z INSDC last update 2017-08-25T17:29:33Z INSDC status public Submitter Id E-MTAB-6018:Able_D4.fastq.gz broker name ArrayExpress cell type neuron common name human disease autism spectrum disorder genotype pRV-CAG:LckN-eGFP individual Able organism part skin phenotype Ngn2+/PSA-NCAM+ progenitor cell type neuronal stem cell sample name E-MTAB-6018:Able_D4.fastq.gz time 4 ERS1886929 SAMEA104227911 9606 Homo sapiens Protocols: 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). ENA first public 2018-01-01 ENA last update 2017-08-25 External Id SAMEA104227911 INSDC center alias Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC center name Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC first public 2018-01-01T17:01:42Z INSDC last update 2017-08-25T17:29:33Z INSDC status public Submitter Id E-MTAB-6018:Able_D7.fastq.gz broker name ArrayExpress cell type neuron common name human disease autism spectrum disorder genotype pRV-CAG:LckN-eGFP individual Able organism part skin phenotype Ngn2+/PSA-NCAM+ progenitor cell type neuronal stem cell sample name E-MTAB-6018:Able_D7.fastq.gz time 7 ERS1886931 SAMEA104227913 9606 Homo sapiens Protocols: 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. Induced iPSC neurons (iPSC-iNs): The cell surface markers SSEA4+ and TRA1-81+ were used for FACS-based purification of iPSCs. For iPSC-iNs, the human cDNA sequence for Ngn2 and the sequence for eGFP were linked by a 2A sequence and cloned into the tet-inducible pLV-XTP backbone (Clonetech). IPSCs were transduced with lentiviral particles for pLV-X-UbC:TetOn (UtO) and pLV-X-Ngn2:2A:eGFP (N2AG) and were selected in the presence of puromycin (0.5 μg/ml, Sigma Aldrich) and G418 (100 μg/ml, Life technologies). For iPSC-iN conversion, iPSCs were transferred into PluriPro monolayer conditions (Cell Guidance Systems) and, 24 hrs after plating, were induced with doxycycline (2 μg/ml: Sigma Aldrich) for two days. For further maturation, iNs were switched to Neuron Maturation Media (NMM) based on DMEM:F12/Neurobasal (both Gibco) (1:1) as used for directed differentiation. NMM contains the following supplements: N2 supplement (1x, Gibco), B27 supplement (1x, Gibco), Laminin (1 μg/ml, Life Technologies) and doxycycline (2 μg/ml: Sigma Aldrich). Medium was changed every other day. Differentiating iPSC-iNs were purified by gating on eGFP+/PSA-NCAM+ neurons at 3, 7 and 14 days after conversion. 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). ENA first public 2018-01-01 ENA last update 2017-08-25 External Id SAMEA104227913 INSDC center alias Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC center name Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC first public 2018-01-01T17:01:42Z INSDC last update 2017-08-25T17:29:33Z INSDC status public Submitter Id E-MTAB-6018:Able_iPSC_iN3d.fastq.gz broker name ArrayExpress cell type neuron common name human disease autism spectrum disorder genotype pLV-X-Ngn2-2A-eGFP (N2AG) individual Able organism part skin phenotype Ngn2+/PSA-NCAM+ progenitor cell type induced pluripotent stem cell sample name E-MTAB-6018:Able_iPSC_iN3d.fastq.gz time 3 ERS1886932 SAMEA104227914 9606 Homo sapiens Protocols: 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. Induced iPSC neurons (iPSC-iNs): The cell surface markers SSEA4+ and TRA1-81+ were used for FACS-based purification of iPSCs. For iPSC-iNs, the human cDNA sequence for Ngn2 and the sequence for eGFP were linked by a 2A sequence and cloned into the tet-inducible pLV-XTP backbone (Clonetech). IPSCs were transduced with lentiviral particles for pLV-X-UbC:TetOn (UtO) and pLV-X-Ngn2:2A:eGFP (N2AG) and were selected in the presence of puromycin (0.5 μg/ml, Sigma Aldrich) and G418 (100 μg/ml, Life technologies). For iPSC-iN conversion, iPSCs were transferred into PluriPro monolayer conditions (Cell Guidance Systems) and, 24 hrs after plating, were induced with doxycycline (2 μg/ml: Sigma Aldrich) for two days. For further maturation, iNs were switched to Neuron Maturation Media (NMM) based on DMEM:F12/Neurobasal (both Gibco) (1:1) as used for directed differentiation. NMM contains the following supplements: N2 supplement (1x, Gibco), B27 supplement (1x, Gibco), Laminin (1 μg/ml, Life Technologies) and doxycycline (2 μg/ml: Sigma Aldrich). Medium was changed every other day. Differentiating iPSC-iNs were purified by gating on eGFP+/PSA-NCAM+ neurons at 3, 7 and 14 days after conversion. 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). ENA first public 2018-01-01 ENA last update 2017-08-25 External Id SAMEA104227914 INSDC center alias Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC center name Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC first public 2018-01-01T17:01:42Z INSDC last update 2017-08-25T17:29:33Z INSDC status public Submitter Id E-MTAB-6018:Able_iPSC_iN7d.fastq.gz broker name ArrayExpress cell type neuron common name human disease autism spectrum disorder genotype pLV-X-Ngn2-2A-eGFP (N2AG) individual Able organism part skin phenotype Ngn2+/PSA-NCAM+ progenitor cell type induced pluripotent stem cell sample name E-MTAB-6018:Able_iPSC_iN7d.fastq.gz time 7 ERS1886933 SAMEA104227915 9606 Homo sapiens Protocols: 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). ENA first public 2018-01-01 ENA last update 2017-08-25 External Id SAMEA104227915 INSDC center alias Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC center name Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC first public 2018-01-01T17:01:42Z INSDC last update 2017-08-25T17:29:33Z INSDC status public Submitter Id E-MTAB-6018:Acai_D14.fastq.gz broker name ArrayExpress cell type neuron common name human disease autism spectrum disorder genotype pRV-CAG:LckN-eGFP individual Acai organism part skin phenotype Ngn2+/PSA-NCAM+ progenitor cell type neuronal stem cell sample name E-MTAB-6018:Acai_D14.fastq.gz time 14 ERS1886934 SAMEA104227916 9606 Homo sapiens Protocols: 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). ENA first public 2018-01-01 ENA last update 2017-08-25 External Id SAMEA104227916 INSDC center alias Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC center name Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC first public 2018-01-01T17:01:42Z INSDC last update 2017-08-25T17:29:33Z INSDC status public Submitter Id E-MTAB-6018:Acai_D2.fastq.gz broker name ArrayExpress cell type neuron common name human disease autism spectrum disorder genotype pRV-CAG:LckN-eGFP individual Acai organism part skin phenotype Ngn2+/PSA-NCAM+ progenitor cell type neuronal stem cell sample name E-MTAB-6018:Acai_D2.fastq.gz time 2 ERS1886935 SAMEA104227917 9606 Homo sapiens Protocols: 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). ENA first public 2018-01-01 ENA last update 2017-08-25 External Id SAMEA104227917 INSDC center alias Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC center name Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC first public 2018-01-01T17:01:42Z INSDC last update 2017-08-25T17:29:33Z INSDC status public Submitter Id E-MTAB-6018:Acai_D4.fastq.gz broker name ArrayExpress cell type neuron common name human disease autism spectrum disorder genotype pRV-CAG:LckN-eGFP individual Acai organism part skin phenotype Ngn2+/PSA-NCAM+ progenitor cell type neuronal stem cell sample name E-MTAB-6018:Acai_D4.fastq.gz time 4 ERS1886936 SAMEA104227918 9606 Homo sapiens Protocols: 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). ENA first public 2018-01-01 ENA last update 2017-08-25 External Id SAMEA104227918 INSDC center alias Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC center name Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC first public 2018-01-01T17:01:42Z INSDC last update 2017-08-25T17:29:33Z INSDC status public Submitter Id E-MTAB-6018:Acai_D7.fastq.gz broker name ArrayExpress cell type neuron common name human disease autism spectrum disorder genotype pRV-CAG:LckN-eGFP individual Acai organism part skin phenotype Ngn2+/PSA-NCAM+ progenitor cell type neuronal stem cell sample name E-MTAB-6018:Acai_D7.fastq.gz time 7 ERS1886937 SAMEA104227919 9606 Homo sapiens Protocols: 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. Induced iPSC neurons (iPSC-iNs): The cell surface markers SSEA4+ and TRA1-81+ were used for FACS-based purification of iPSCs. For iPSC-iNs, the human cDNA sequence for Ngn2 and the sequence for eGFP were linked by a 2A sequence and cloned into the tet-inducible pLV-XTP backbone (Clonetech). IPSCs were transduced with lentiviral particles for pLV-X-UbC:TetOn (UtO) and pLV-X-Ngn2:2A:eGFP (N2AG) and were selected in the presence of puromycin (0.5 μg/ml, Sigma Aldrich) and G418 (100 μg/ml, Life technologies). For iPSC-iN conversion, iPSCs were transferred into PluriPro monolayer conditions (Cell Guidance Systems) and, 24 hrs after plating, were induced with doxycycline (2 μg/ml: Sigma Aldrich) for two days. For further maturation, iNs were switched to Neuron Maturation Media (NMM) based on DMEM:F12/Neurobasal (both Gibco) (1:1) as used for directed differentiation. NMM contains the following supplements: N2 supplement (1x, Gibco), B27 supplement (1x, Gibco), Laminin (1 μg/ml, Life Technologies) and doxycycline (2 μg/ml: Sigma Aldrich). Medium was changed every other day. Differentiating iPSC-iNs were purified by gating on eGFP+/PSA-NCAM+ neurons at 3, 7 and 14 days after conversion. 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). ENA first public 2018-01-01 ENA last update 2017-08-25 External Id SAMEA104227919 INSDC center alias Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC center name Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC first public 2018-01-01T17:01:42Z INSDC last update 2017-08-25T17:29:33Z INSDC status public Submitter Id E-MTAB-6018:Acai_iPSC_iN14d.fastq.gz broker name ArrayExpress cell type neuron common name human disease autism spectrum disorder genotype pLV-X-Ngn2-2A-eGFP (N2AG) individual Acai organism part skin phenotype Ngn2+/PSA-NCAM+ progenitor cell type induced pluripotent stem cell sample name E-MTAB-6018:Acai_iPSC_iN14d.fastq.gz time 14 ERS1886938 SAMEA104227920 9606 Homo sapiens Protocols: 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. Induced iPSC neurons (iPSC-iNs): The cell surface markers SSEA4+ and TRA1-81+ were used for FACS-based purification of iPSCs. For iPSC-iNs, the human cDNA sequence for Ngn2 and the sequence for eGFP were linked by a 2A sequence and cloned into the tet-inducible pLV-XTP backbone (Clonetech). IPSCs were transduced with lentiviral particles for pLV-X-UbC:TetOn (UtO) and pLV-X-Ngn2:2A:eGFP (N2AG) and were selected in the presence of puromycin (0.5 μg/ml, Sigma Aldrich) and G418 (100 μg/ml, Life technologies). For iPSC-iN conversion, iPSCs were transferred into PluriPro monolayer conditions (Cell Guidance Systems) and, 24 hrs after plating, were induced with doxycycline (2 μg/ml: Sigma Aldrich) for two days. For further maturation, iNs were switched to Neuron Maturation Media (NMM) based on DMEM:F12/Neurobasal (both Gibco) (1:1) as used for directed differentiation. NMM contains the following supplements: N2 supplement (1x, Gibco), B27 supplement (1x, Gibco), Laminin (1 μg/ml, Life Technologies) and doxycycline (2 μg/ml: Sigma Aldrich). Medium was changed every other day. Differentiating iPSC-iNs were purified by gating on eGFP+/PSA-NCAM+ neurons at 3, 7 and 14 days after conversion. 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). ENA first public 2018-01-01 ENA last update 2017-08-25 External Id SAMEA104227920 INSDC center alias Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC center name Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC first public 2018-01-01T17:01:42Z INSDC last update 2017-08-25T17:29:33Z INSDC status public Submitter Id E-MTAB-6018:Acai_iPSC_iN3d.fastq.gz broker name ArrayExpress cell type neuron common name human disease autism spectrum disorder genotype pLV-X-Ngn2-2A-eGFP (N2AG) individual Acai organism part skin phenotype Ngn2+/PSA-NCAM+ progenitor cell type induced pluripotent stem cell sample name E-MTAB-6018:Acai_iPSC_iN3d.fastq.gz time 3 ERS1886939 SAMEA104227921 9606 Homo sapiens Protocols: 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. Induced iPSC neurons (iPSC-iNs): The cell surface markers SSEA4+ and TRA1-81+ were used for FACS-based purification of iPSCs. For iPSC-iNs, the human cDNA sequence for Ngn2 and the sequence for eGFP were linked by a 2A sequence and cloned into the tet-inducible pLV-XTP backbone (Clonetech). IPSCs were transduced with lentiviral particles for pLV-X-UbC:TetOn (UtO) and pLV-X-Ngn2:2A:eGFP (N2AG) and were selected in the presence of puromycin (0.5 μg/ml, Sigma Aldrich) and G418 (100 μg/ml, Life technologies). For iPSC-iN conversion, iPSCs were transferred into PluriPro monolayer conditions (Cell Guidance Systems) and, 24 hrs after plating, were induced with doxycycline (2 μg/ml: Sigma Aldrich) for two days. For further maturation, iNs were switched to Neuron Maturation Media (NMM) based on DMEM:F12/Neurobasal (both Gibco) (1:1) as used for directed differentiation. NMM contains the following supplements: N2 supplement (1x, Gibco), B27 supplement (1x, Gibco), Laminin (1 μg/ml, Life Technologies) and doxycycline (2 μg/ml: Sigma Aldrich). Medium was changed every other day. Differentiating iPSC-iNs were purified by gating on eGFP+/PSA-NCAM+ neurons at 3, 7 and 14 days after conversion. 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). ENA first public 2018-01-01 ENA last update 2017-08-25 External Id SAMEA104227921 INSDC center alias Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC center name Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC first public 2018-01-01T17:01:42Z INSDC last update 2017-08-25T17:29:33Z INSDC status public Submitter Id E-MTAB-6018:Acai_iPSC_iN7d.fastq.gz broker name ArrayExpress cell type neuron common name human disease autism spectrum disorder genotype pLV-X-Ngn2-2A-eGFP (N2AG) individual Acai organism part skin phenotype Ngn2+/PSA-NCAM+ progenitor cell type induced pluripotent stem cell sample name E-MTAB-6018:Acai_iPSC_iN7d.fastq.gz time 7 ERS1886940 SAMEA104227922 9606 Homo sapiens Protocols: 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). ENA first public 2018-01-01 ENA last update 2017-08-25 External Id SAMEA104227922 INSDC center alias Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC center name Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC first public 2018-01-01T17:01:42Z INSDC last update 2017-08-25T17:29:33Z INSDC status public Submitter Id E-MTAB-6018:Aero_D14.fastq.gz broker name ArrayExpress cell type neuron common name human disease autism spectrum disorder genotype pRV-CAG:LckN-eGFP individual Aero organism part skin phenotype Ngn2+/PSA-NCAM+ progenitor cell type neuronal stem cell sample name E-MTAB-6018:Aero_D14.fastq.gz time 14 ERS1886941 SAMEA104227923 9606 Homo sapiens Protocols: 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). ENA first public 2018-01-01 ENA last update 2017-08-25 External Id SAMEA104227923 INSDC center alias Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC center name Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC first public 2018-01-01T17:01:42Z INSDC last update 2017-08-25T17:29:33Z INSDC status public Submitter Id E-MTAB-6018:Aero_D2.fastq.gz broker name ArrayExpress cell type neuron common name human disease autism spectrum disorder genotype pRV-CAG:LckN-eGFP individual Aero organism part skin phenotype Ngn2+/PSA-NCAM+ progenitor cell type neuronal stem cell sample name E-MTAB-6018:Aero_D2.fastq.gz time 2 ERS1886942 SAMEA104227924 9606 Homo sapiens Protocols: 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). ENA first public 2018-01-01 ENA last update 2017-08-25 External Id SAMEA104227924 INSDC center alias Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC center name Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC first public 2018-01-01T17:01:42Z INSDC last update 2017-08-25T17:29:33Z INSDC status public Submitter Id E-MTAB-6018:Aero_D4.fastq.gz broker name ArrayExpress cell type neuron common name human disease autism spectrum disorder genotype pRV-CAG:LckN-eGFP individual Aero organism part skin phenotype Ngn2+/PSA-NCAM+ progenitor cell type neuronal stem cell sample name E-MTAB-6018:Aero_D4.fastq.gz time 4 ERS1886943 SAMEA104227925 9606 Homo sapiens Protocols: 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). ENA first public 2018-01-01 ENA last update 2017-08-25 External Id SAMEA104227925 INSDC center alias Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC center name Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC first public 2018-01-01T17:01:42Z INSDC last update 2017-08-25T17:29:33Z INSDC status public Submitter Id E-MTAB-6018:Aero_D7.fastq.gz broker name ArrayExpress cell type neuron common name human disease autism spectrum disorder genotype pRV-CAG:LckN-eGFP individual Aero organism part skin phenotype Ngn2+/PSA-NCAM+ progenitor cell type neuronal stem cell sample name E-MTAB-6018:Aero_D7.fastq.gz time 7 ERS1886944 SAMEA104227926 9606 Homo sapiens Protocols: 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. Induced iPSC neurons (iPSC-iNs): The cell surface markers SSEA4+ and TRA1-81+ were used for FACS-based purification of iPSCs. For iPSC-iNs, the human cDNA sequence for Ngn2 and the sequence for eGFP were linked by a 2A sequence and cloned into the tet-inducible pLV-XTP backbone (Clonetech). IPSCs were transduced with lentiviral particles for pLV-X-UbC:TetOn (UtO) and pLV-X-Ngn2:2A:eGFP (N2AG) and were selected in the presence of puromycin (0.5 μg/ml, Sigma Aldrich) and G418 (100 μg/ml, Life technologies). For iPSC-iN conversion, iPSCs were transferred into PluriPro monolayer conditions (Cell Guidance Systems) and, 24 hrs after plating, were induced with doxycycline (2 μg/ml: Sigma Aldrich) for two days. For further maturation, iNs were switched to Neuron Maturation Media (NMM) based on DMEM:F12/Neurobasal (both Gibco) (1:1) as used for directed differentiation. NMM contains the following supplements: N2 supplement (1x, Gibco), B27 supplement (1x, Gibco), Laminin (1 μg/ml, Life Technologies) and doxycycline (2 μg/ml: Sigma Aldrich). Medium was changed every other day. Differentiating iPSC-iNs were purified by gating on eGFP+/PSA-NCAM+ neurons at 3, 7 and 14 days after conversion. 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). ENA first public 2018-01-01 ENA last update 2017-08-25 External Id SAMEA104227926 INSDC center alias Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC center name Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC first public 2018-01-01T17:01:42Z INSDC last update 2017-08-25T17:29:33Z INSDC status public Submitter Id E-MTAB-6018:Aero_iPSC_iN14d.fastq.gz broker name ArrayExpress cell type neuron common name human disease autism spectrum disorder genotype pLV-X-Ngn2-2A-eGFP (N2AG) individual Aero organism part skin phenotype Ngn2+/PSA-NCAM+ progenitor cell type induced pluripotent stem cell sample name E-MTAB-6018:Aero_iPSC_iN14d.fastq.gz time 14 ERS1886945 SAMEA104227927 9606 Homo sapiens Protocols: 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. Induced iPSC neurons (iPSC-iNs): The cell surface markers SSEA4+ and TRA1-81+ were used for FACS-based purification of iPSCs. For iPSC-iNs, the human cDNA sequence for Ngn2 and the sequence for eGFP were linked by a 2A sequence and cloned into the tet-inducible pLV-XTP backbone (Clonetech). IPSCs were transduced with lentiviral particles for pLV-X-UbC:TetOn (UtO) and pLV-X-Ngn2:2A:eGFP (N2AG) and were selected in the presence of puromycin (0.5 μg/ml, Sigma Aldrich) and G418 (100 μg/ml, Life technologies). For iPSC-iN conversion, iPSCs were transferred into PluriPro monolayer conditions (Cell Guidance Systems) and, 24 hrs after plating, were induced with doxycycline (2 μg/ml: Sigma Aldrich) for two days. For further maturation, iNs were switched to Neuron Maturation Media (NMM) based on DMEM:F12/Neurobasal (both Gibco) (1:1) as used for directed differentiation. NMM contains the following supplements: N2 supplement (1x, Gibco), B27 supplement (1x, Gibco), Laminin (1 μg/ml, Life Technologies) and doxycycline (2 μg/ml: Sigma Aldrich). Medium was changed every other day. Differentiating iPSC-iNs were purified by gating on eGFP+/PSA-NCAM+ neurons at 3, 7 and 14 days after conversion. 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). ENA first public 2018-01-01 ENA last update 2017-08-25 External Id SAMEA104227927 INSDC center alias Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC center name Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC first public 2018-01-01T17:01:42Z INSDC last update 2017-08-25T17:29:33Z INSDC status public Submitter Id E-MTAB-6018:Aero_iPSC_iN3d.fastq.gz broker name ArrayExpress cell type neuron common name human disease autism spectrum disorder genotype pLV-X-Ngn2-2A-eGFP (N2AG) individual Aero organism part skin phenotype Ngn2+/PSA-NCAM+ progenitor cell type induced pluripotent stem cell sample name E-MTAB-6018:Aero_iPSC_iN3d.fastq.gz time 3 ERS1886946 SAMEA104227928 9606 Homo sapiens Protocols: 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. Induced iPSC neurons (iPSC-iNs): The cell surface markers SSEA4+ and TRA1-81+ were used for FACS-based purification of iPSCs. For iPSC-iNs, the human cDNA sequence for Ngn2 and the sequence for eGFP were linked by a 2A sequence and cloned into the tet-inducible pLV-XTP backbone (Clonetech). IPSCs were transduced with lentiviral particles for pLV-X-UbC:TetOn (UtO) and pLV-X-Ngn2:2A:eGFP (N2AG) and were selected in the presence of puromycin (0.5 μg/ml, Sigma Aldrich) and G418 (100 μg/ml, Life technologies). For iPSC-iN conversion, iPSCs were transferred into PluriPro monolayer conditions (Cell Guidance Systems) and, 24 hrs after plating, were induced with doxycycline (2 μg/ml: Sigma Aldrich) for two days. For further maturation, iNs were switched to Neuron Maturation Media (NMM) based on DMEM:F12/Neurobasal (both Gibco) (1:1) as used for directed differentiation. NMM contains the following supplements: N2 supplement (1x, Gibco), B27 supplement (1x, Gibco), Laminin (1 μg/ml, Life Technologies) and doxycycline (2 μg/ml: Sigma Aldrich). Medium was changed every other day. Differentiating iPSC-iNs were purified by gating on eGFP+/PSA-NCAM+ neurons at 3, 7 and 14 days after conversion. 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). ENA first public 2018-01-01 ENA last update 2017-08-25 External Id SAMEA104227928 INSDC center alias Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC center name Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC first public 2018-01-01T17:01:42Z INSDC last update 2017-08-25T17:29:33Z INSDC status public Submitter Id E-MTAB-6018:Aero_iPSC_iN7d.fastq.gz broker name ArrayExpress cell type neuron common name human disease autism spectrum disorder genotype pLV-X-Ngn2-2A-eGFP (N2AG) individual Aero organism part skin phenotype Ngn2+/PSA-NCAM+ progenitor cell type induced pluripotent stem cell sample name E-MTAB-6018:Aero_iPSC_iN7d.fastq.gz time 7 ERS1886947 SAMEA104227929 9606 Homo sapiens Protocols: 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). ENA first public 2018-01-01 ENA last update 2017-08-25 External Id SAMEA104227929 INSDC center alias Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC center name Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC first public 2018-01-01T17:01:42Z INSDC last update 2017-08-25T17:29:33Z INSDC status public Submitter Id E-MTAB-6018:Agua_D14.fastq.gz broker name ArrayExpress cell type neuron common name human disease autism spectrum disorder genotype pRV-CAG:LckN-eGFP individual Agua organism part skin phenotype Ngn2+/PSA-NCAM+ progenitor cell type neuronal stem cell sample name E-MTAB-6018:Agua_D14.fastq.gz time 14 ERS1886948 SAMEA104227930 9606 Homo sapiens Protocols: 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). ENA first public 2018-01-01 ENA last update 2017-08-25 External Id SAMEA104227930 INSDC center alias Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC center name Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC first public 2018-01-01T17:01:42Z INSDC last update 2017-08-25T17:29:34Z INSDC status public Submitter Id E-MTAB-6018:Agua_D2.fastq.gz broker name ArrayExpress cell type neuron common name human disease autism spectrum disorder genotype pRV-CAG:LckN-eGFP individual Agua organism part skin phenotype Ngn2+/PSA-NCAM+ progenitor cell type neuronal stem cell sample name E-MTAB-6018:Agua_D2.fastq.gz time 2 ERS1886949 SAMEA104227931 9606 Homo sapiens Protocols: 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). ENA first public 2018-01-01 ENA last update 2017-08-25 External Id SAMEA104227931 INSDC center alias Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC center name Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC first public 2018-01-01T17:01:42Z INSDC last update 2017-08-25T17:29:34Z INSDC status public Submitter Id E-MTAB-6018:Agua_D4.fastq.gz broker name ArrayExpress cell type neuron common name human disease autism spectrum disorder genotype pRV-CAG:LckN-eGFP individual Agua organism part skin phenotype Ngn2+/PSA-NCAM+ progenitor cell type neuronal stem cell sample name E-MTAB-6018:Agua_D4.fastq.gz time 4 ERS1886950 SAMEA104227932 9606 Homo sapiens Protocols: 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). ENA first public 2018-01-01 ENA last update 2017-08-25 External Id SAMEA104227932 INSDC center alias Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC center name Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC first public 2018-01-01T17:01:42Z INSDC last update 2017-08-25T17:29:34Z INSDC status public Submitter Id E-MTAB-6018:Agua_D7.fastq.gz broker name ArrayExpress cell type neuron common name human disease autism spectrum disorder genotype pRV-CAG:LckN-eGFP individual Agua organism part skin phenotype Ngn2+/PSA-NCAM+ progenitor cell type neuronal stem cell sample name E-MTAB-6018:Agua_D7.fastq.gz time 7 ERS1886951 SAMEA104227933 9606 Homo sapiens Protocols: 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. Induced iPSC neurons (iPSC-iNs): The cell surface markers SSEA4+ and TRA1-81+ were used for FACS-based purification of iPSCs. For iPSC-iNs, the human cDNA sequence for Ngn2 and the sequence for eGFP were linked by a 2A sequence and cloned into the tet-inducible pLV-XTP backbone (Clonetech). IPSCs were transduced with lentiviral particles for pLV-X-UbC:TetOn (UtO) and pLV-X-Ngn2:2A:eGFP (N2AG) and were selected in the presence of puromycin (0.5 μg/ml, Sigma Aldrich) and G418 (100 μg/ml, Life technologies). For iPSC-iN conversion, iPSCs were transferred into PluriPro monolayer conditions (Cell Guidance Systems) and, 24 hrs after plating, were induced with doxycycline (2 μg/ml: Sigma Aldrich) for two days. For further maturation, iNs were switched to Neuron Maturation Media (NMM) based on DMEM:F12/Neurobasal (both Gibco) (1:1) as used for directed differentiation. NMM contains the following supplements: N2 supplement (1x, Gibco), B27 supplement (1x, Gibco), Laminin (1 μg/ml, Life Technologies) and doxycycline (2 μg/ml: Sigma Aldrich). Medium was changed every other day. Differentiating iPSC-iNs were purified by gating on eGFP+/PSA-NCAM+ neurons at 3, 7 and 14 days after conversion. 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). ENA first public 2018-01-01 ENA last update 2017-08-25 External Id SAMEA104227933 INSDC center alias Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC center name Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC first public 2018-01-01T17:01:42Z INSDC last update 2017-08-25T17:29:34Z INSDC status public Submitter Id E-MTAB-6018:Agua_iPSC_iN14d.fastq.gz broker name ArrayExpress cell type neuron common name human disease autism spectrum disorder genotype pLV-X-Ngn2-2A-eGFP (N2AG) individual Agua organism part skin phenotype Ngn2+/PSA-NCAM+ progenitor cell type induced pluripotent stem cell sample name E-MTAB-6018:Agua_iPSC_iN14d.fastq.gz time 14 ERS1886952 SAMEA104227934 9606 Homo sapiens Protocols: 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. Induced iPSC neurons (iPSC-iNs): The cell surface markers SSEA4+ and TRA1-81+ were used for FACS-based purification of iPSCs. For iPSC-iNs, the human cDNA sequence for Ngn2 and the sequence for eGFP were linked by a 2A sequence and cloned into the tet-inducible pLV-XTP backbone (Clonetech). IPSCs were transduced with lentiviral particles for pLV-X-UbC:TetOn (UtO) and pLV-X-Ngn2:2A:eGFP (N2AG) and were selected in the presence of puromycin (0.5 μg/ml, Sigma Aldrich) and G418 (100 μg/ml, Life technologies). For iPSC-iN conversion, iPSCs were transferred into PluriPro monolayer conditions (Cell Guidance Systems) and, 24 hrs after plating, were induced with doxycycline (2 μg/ml: Sigma Aldrich) for two days. For further maturation, iNs were switched to Neuron Maturation Media (NMM) based on DMEM:F12/Neurobasal (both Gibco) (1:1) as used for directed differentiation. NMM contains the following supplements: N2 supplement (1x, Gibco), B27 supplement (1x, Gibco), Laminin (1 μg/ml, Life Technologies) and doxycycline (2 μg/ml: Sigma Aldrich). Medium was changed every other day. Differentiating iPSC-iNs were purified by gating on eGFP+/PSA-NCAM+ neurons at 3, 7 and 14 days after conversion. 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). ENA first public 2018-01-01 ENA last update 2017-08-25 External Id SAMEA104227934 INSDC center alias Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC center name Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC first public 2018-01-01T17:01:42Z INSDC last update 2017-08-25T17:29:34Z INSDC status public Submitter Id E-MTAB-6018:Agua_iPSC_iN3d.fastq.gz broker name ArrayExpress cell type neuron common name human disease autism spectrum disorder genotype pLV-X-Ngn2-2A-eGFP (N2AG) individual Agua organism part skin phenotype Ngn2+/PSA-NCAM+ progenitor cell type induced pluripotent stem cell sample name E-MTAB-6018:Agua_iPSC_iN3d.fastq.gz time 3 ERS1886953 SAMEA104227935 9606 Homo sapiens Protocols: 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. Induced iPSC neurons (iPSC-iNs): The cell surface markers SSEA4+ and TRA1-81+ were used for FACS-based purification of iPSCs. For iPSC-iNs, the human cDNA sequence for Ngn2 and the sequence for eGFP were linked by a 2A sequence and cloned into the tet-inducible pLV-XTP backbone (Clonetech). IPSCs were transduced with lentiviral particles for pLV-X-UbC:TetOn (UtO) and pLV-X-Ngn2:2A:eGFP (N2AG) and were selected in the presence of puromycin (0.5 μg/ml, Sigma Aldrich) and G418 (100 μg/ml, Life technologies). For iPSC-iN conversion, iPSCs were transferred into PluriPro monolayer conditions (Cell Guidance Systems) and, 24 hrs after plating, were induced with doxycycline (2 μg/ml: Sigma Aldrich) for two days. For further maturation, iNs were switched to Neuron Maturation Media (NMM) based on DMEM:F12/Neurobasal (both Gibco) (1:1) as used for directed differentiation. NMM contains the following supplements: N2 supplement (1x, Gibco), B27 supplement (1x, Gibco), Laminin (1 μg/ml, Life Technologies) and doxycycline (2 μg/ml: Sigma Aldrich). Medium was changed every other day. Differentiating iPSC-iNs were purified by gating on eGFP+/PSA-NCAM+ neurons at 3, 7 and 14 days after conversion. 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). ENA first public 2018-01-01 ENA last update 2017-08-25 External Id SAMEA104227935 INSDC center alias Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC center name Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC first public 2018-01-01T17:01:42Z INSDC last update 2017-08-25T17:29:34Z INSDC status public Submitter Id E-MTAB-6018:Agua_iPSC_iN7d.fastq.gz broker name ArrayExpress cell type neuron common name human disease autism spectrum disorder genotype pLV-X-Ngn2-2A-eGFP (N2AG) individual Agua organism part skin phenotype Ngn2+/PSA-NCAM+ progenitor cell type induced pluripotent stem cell sample name E-MTAB-6018:Agua_iPSC_iN7d.fastq.gz time 7 ERS1886954 SAMEA104227936 9606 Homo sapiens Protocols: 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). ENA first public 2018-01-01 ENA last update 2017-08-25 External Id SAMEA104227936 INSDC center alias Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC center name Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC first public 2018-01-01T17:01:42Z INSDC last update 2017-08-25T17:29:34Z INSDC status public Submitter Id E-MTAB-6018:Ahoy_D14.fastq.gz broker name ArrayExpress cell type neuron common name human disease autism spectrum disorder genotype pRV-CAG:LckN-eGFP individual Ahoy organism part skin phenotype Ngn2+/PSA-NCAM+ progenitor cell type neuronal stem cell sample name E-MTAB-6018:Ahoy_D14.fastq.gz time 14 ERS1886955 SAMEA104227937 9606 Homo sapiens Protocols: 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). ENA first public 2018-01-01 ENA last update 2017-08-25 External Id SAMEA104227937 INSDC center alias Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC center name Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC first public 2018-01-01T17:01:42Z INSDC last update 2017-08-25T17:29:34Z INSDC status public Submitter Id E-MTAB-6018:Ahoy_D2.fastq.gz broker name ArrayExpress cell type neuron common name human disease autism spectrum disorder genotype pRV-CAG:LckN-eGFP individual Ahoy organism part skin phenotype Ngn2+/PSA-NCAM+ progenitor cell type neuronal stem cell sample name E-MTAB-6018:Ahoy_D2.fastq.gz time 2 ERS1886956 SAMEA104227938 9606 Homo sapiens Protocols: 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). ENA first public 2018-01-01 ENA last update 2017-08-25 External Id SAMEA104227938 INSDC center alias Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC center name Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC first public 2018-01-01T17:01:42Z INSDC last update 2017-08-25T17:29:34Z INSDC status public Submitter Id E-MTAB-6018:Ahoy_D4.fastq.gz broker name ArrayExpress cell type neuron common name human disease autism spectrum disorder genotype pRV-CAG:LckN-eGFP individual Ahoy organism part skin phenotype Ngn2+/PSA-NCAM+ progenitor cell type neuronal stem cell sample name E-MTAB-6018:Ahoy_D4.fastq.gz time 4 ERS1886957 SAMEA104227939 9606 Homo sapiens Protocols: 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). ENA first public 2018-01-01 ENA last update 2017-08-25 External Id SAMEA104227939 INSDC center alias Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC center name Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC first public 2018-01-01T17:01:42Z INSDC last update 2017-08-25T17:29:34Z INSDC status public Submitter Id E-MTAB-6018:Ahoy_D7.fastq.gz broker name ArrayExpress cell type neuron common name human disease autism spectrum disorder genotype pRV-CAG:LckN-eGFP individual Ahoy organism part skin phenotype Ngn2+/PSA-NCAM+ progenitor cell type neuronal stem cell sample name E-MTAB-6018:Ahoy_D7.fastq.gz time 7 ERS1886958 SAMEA104227940 9606 Homo sapiens Protocols: 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. Induced iPSC neurons (iPSC-iNs): The cell surface markers SSEA4+ and TRA1-81+ were used for FACS-based purification of iPSCs. For iPSC-iNs, the human cDNA sequence for Ngn2 and the sequence for eGFP were linked by a 2A sequence and cloned into the tet-inducible pLV-XTP backbone (Clonetech). IPSCs were transduced with lentiviral particles for pLV-X-UbC:TetOn (UtO) and pLV-X-Ngn2:2A:eGFP (N2AG) and were selected in the presence of puromycin (0.5 μg/ml, Sigma Aldrich) and G418 (100 μg/ml, Life technologies). For iPSC-iN conversion, iPSCs were transferred into PluriPro monolayer conditions (Cell Guidance Systems) and, 24 hrs after plating, were induced with doxycycline (2 μg/ml: Sigma Aldrich) for two days. For further maturation, iNs were switched to Neuron Maturation Media (NMM) based on DMEM:F12/Neurobasal (both Gibco) (1:1) as used for directed differentiation. NMM contains the following supplements: N2 supplement (1x, Gibco), B27 supplement (1x, Gibco), Laminin (1 μg/ml, Life Technologies) and doxycycline (2 μg/ml: Sigma Aldrich). Medium was changed every other day. Differentiating iPSC-iNs were purified by gating on eGFP+/PSA-NCAM+ neurons at 3, 7 and 14 days after conversion. 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). ENA first public 2018-01-01 ENA last update 2017-08-25 External Id SAMEA104227940 INSDC center alias Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC center name Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC first public 2018-01-01T17:01:42Z INSDC last update 2017-08-25T17:29:34Z INSDC status public Submitter Id E-MTAB-6018:Ahoy_iPSC_iN14d.fastq.gz broker name ArrayExpress cell type neuron common name human disease autism spectrum disorder genotype pLV-X-Ngn2-2A-eGFP (N2AG) individual Ahoy organism part skin phenotype Ngn2+/PSA-NCAM+ progenitor cell type induced pluripotent stem cell sample name E-MTAB-6018:Ahoy_iPSC_iN14d.fastq.gz time 14 ERS1886959 SAMEA104227941 9606 Homo sapiens Protocols: 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. Induced iPSC neurons (iPSC-iNs): The cell surface markers SSEA4+ and TRA1-81+ were used for FACS-based purification of iPSCs. For iPSC-iNs, the human cDNA sequence for Ngn2 and the sequence for eGFP were linked by a 2A sequence and cloned into the tet-inducible pLV-XTP backbone (Clonetech). IPSCs were transduced with lentiviral particles for pLV-X-UbC:TetOn (UtO) and pLV-X-Ngn2:2A:eGFP (N2AG) and were selected in the presence of puromycin (0.5 μg/ml, Sigma Aldrich) and G418 (100 μg/ml, Life technologies). For iPSC-iN conversion, iPSCs were transferred into PluriPro monolayer conditions (Cell Guidance Systems) and, 24 hrs after plating, were induced with doxycycline (2 μg/ml: Sigma Aldrich) for two days. For further maturation, iNs were switched to Neuron Maturation Media (NMM) based on DMEM:F12/Neurobasal (both Gibco) (1:1) as used for directed differentiation. NMM contains the following supplements: N2 supplement (1x, Gibco), B27 supplement (1x, Gibco), Laminin (1 μg/ml, Life Technologies) and doxycycline (2 μg/ml: Sigma Aldrich). Medium was changed every other day. Differentiating iPSC-iNs were purified by gating on eGFP+/PSA-NCAM+ neurons at 3, 7 and 14 days after conversion. 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). ENA first public 2018-01-01 ENA last update 2017-08-25 External Id SAMEA104227941 INSDC center alias Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC center name Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC first public 2018-01-01T17:01:42Z INSDC last update 2017-08-25T17:29:34Z INSDC status public Submitter Id E-MTAB-6018:Ahoy_iPSC_iN3d.fastq.gz broker name ArrayExpress cell type neuron common name human disease autism spectrum disorder genotype pLV-X-Ngn2-2A-eGFP (N2AG) individual Ahoy organism part skin phenotype Ngn2+/PSA-NCAM+ progenitor cell type induced pluripotent stem cell sample name E-MTAB-6018:Ahoy_iPSC_iN3d.fastq.gz time 3 ERS1886960 SAMEA104227942 9606 Homo sapiens Protocols: 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. Induced iPSC neurons (iPSC-iNs): The cell surface markers SSEA4+ and TRA1-81+ were used for FACS-based purification of iPSCs. For iPSC-iNs, the human cDNA sequence for Ngn2 and the sequence for eGFP were linked by a 2A sequence and cloned into the tet-inducible pLV-XTP backbone (Clonetech). IPSCs were transduced with lentiviral particles for pLV-X-UbC:TetOn (UtO) and pLV-X-Ngn2:2A:eGFP (N2AG) and were selected in the presence of puromycin (0.5 μg/ml, Sigma Aldrich) and G418 (100 μg/ml, Life technologies). For iPSC-iN conversion, iPSCs were transferred into PluriPro monolayer conditions (Cell Guidance Systems) and, 24 hrs after plating, were induced with doxycycline (2 μg/ml: Sigma Aldrich) for two days. For further maturation, iNs were switched to Neuron Maturation Media (NMM) based on DMEM:F12/Neurobasal (both Gibco) (1:1) as used for directed differentiation. NMM contains the following supplements: N2 supplement (1x, Gibco), B27 supplement (1x, Gibco), Laminin (1 μg/ml, Life Technologies) and doxycycline (2 μg/ml: Sigma Aldrich). Medium was changed every other day. Differentiating iPSC-iNs were purified by gating on eGFP+/PSA-NCAM+ neurons at 3, 7 and 14 days after conversion. 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). ENA first public 2018-01-01 ENA last update 2017-08-25 External Id SAMEA104227942 INSDC center alias Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC center name Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC first public 2018-01-01T17:01:42Z INSDC last update 2017-08-25T17:29:34Z INSDC status public Submitter Id E-MTAB-6018:Ahoy_iPSC_iN7d.fastq.gz broker name ArrayExpress cell type neuron common name human disease autism spectrum disorder genotype pLV-X-Ngn2-2A-eGFP (N2AG) individual Ahoy organism part skin phenotype Ngn2+/PSA-NCAM+ progenitor cell type induced pluripotent stem cell sample name E-MTAB-6018:Ahoy_iPSC_iN7d.fastq.gz time 7 ERS1886961 SAMEA104227943 9606 Homo sapiens Protocols: 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). ENA first public 2018-01-01 ENA last update 2017-08-25 External Id SAMEA104227943 INSDC center alias Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC center name Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC first public 2018-01-01T17:01:42Z INSDC last update 2017-08-25T17:29:34Z INSDC status public Submitter Id E-MTAB-6018:Apex_D14.fastq.gz broker name ArrayExpress cell type neuron common name human disease autism spectrum disorder genotype pRV-CAG:LckN-eGFP individual Apex organism part skin phenotype Ngn2+/PSA-NCAM+ progenitor cell type neuronal stem cell sample name E-MTAB-6018:Apex_D14.fastq.gz time 14 ERS1886962 SAMEA104227944 9606 Homo sapiens Protocols: 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). ENA first public 2018-01-01 ENA last update 2017-08-25 External Id SAMEA104227944 INSDC center alias Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC center name Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC first public 2018-01-01T17:01:42Z INSDC last update 2017-08-25T17:29:34Z INSDC status public Submitter Id E-MTAB-6018:Apex_D2.fastq.gz broker name ArrayExpress cell type neuron common name human disease autism spectrum disorder genotype pRV-CAG:LckN-eGFP individual Apex organism part skin phenotype Ngn2+/PSA-NCAM+ progenitor cell type neuronal stem cell sample name E-MTAB-6018:Apex_D2.fastq.gz time 2 ERS1886963 SAMEA104227945 9606 Homo sapiens Protocols: 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). ENA first public 2018-01-01 ENA last update 2017-08-25 External Id SAMEA104227945 INSDC center alias Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC center name Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC first public 2018-01-01T17:01:42Z INSDC last update 2017-08-25T17:29:34Z INSDC status public Submitter Id E-MTAB-6018:Apex_D4.fastq.gz broker name ArrayExpress cell type neuron common name human disease autism spectrum disorder genotype pRV-CAG:LckN-eGFP individual Apex organism part skin phenotype Ngn2+/PSA-NCAM+ progenitor cell type neuronal stem cell sample name E-MTAB-6018:Apex_D4.fastq.gz time 4 ERS1886964 SAMEA104227946 9606 Homo sapiens Protocols: 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). ENA first public 2018-01-01 ENA last update 2017-08-25 External Id SAMEA104227946 INSDC center alias Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC center name Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC first public 2018-01-01T17:01:42Z INSDC last update 2017-08-25T17:29:34Z INSDC status public Submitter Id E-MTAB-6018:Apex_D7.fastq.gz broker name ArrayExpress cell type neuron common name human disease autism spectrum disorder genotype pRV-CAG:LckN-eGFP individual Apex organism part skin phenotype Ngn2+/PSA-NCAM+ progenitor cell type neuronal stem cell sample name E-MTAB-6018:Apex_D7.fastq.gz time 7 ERS1886965 SAMEA104227947 9606 Homo sapiens Protocols: 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. Induced iPSC neurons (iPSC-iNs): The cell surface markers SSEA4+ and TRA1-81+ were used for FACS-based purification of iPSCs. For iPSC-iNs, the human cDNA sequence for Ngn2 and the sequence for eGFP were linked by a 2A sequence and cloned into the tet-inducible pLV-XTP backbone (Clonetech). IPSCs were transduced with lentiviral particles for pLV-X-UbC:TetOn (UtO) and pLV-X-Ngn2:2A:eGFP (N2AG) and were selected in the presence of puromycin (0.5 μg/ml, Sigma Aldrich) and G418 (100 μg/ml, Life technologies). For iPSC-iN conversion, iPSCs were transferred into PluriPro monolayer conditions (Cell Guidance Systems) and, 24 hrs after plating, were induced with doxycycline (2 μg/ml: Sigma Aldrich) for two days. For further maturation, iNs were switched to Neuron Maturation Media (NMM) based on DMEM:F12/Neurobasal (both Gibco) (1:1) as used for directed differentiation. NMM contains the following supplements: N2 supplement (1x, Gibco), B27 supplement (1x, Gibco), Laminin (1 μg/ml, Life Technologies) and doxycycline (2 μg/ml: Sigma Aldrich). Medium was changed every other day. Differentiating iPSC-iNs were purified by gating on eGFP+/PSA-NCAM+ neurons at 3, 7 and 14 days after conversion. 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). ENA first public 2018-01-01 ENA last update 2017-08-25 External Id SAMEA104227947 INSDC center alias Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC center name Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC first public 2018-01-01T17:01:42Z INSDC last update 2017-08-25T17:29:34Z INSDC status public Submitter Id E-MTAB-6018:Apex_iPSC_iN14d.fastq.gz broker name ArrayExpress cell type neuron common name human disease autism spectrum disorder genotype pLV-X-Ngn2-2A-eGFP (N2AG) individual Apex organism part skin phenotype Ngn2+/PSA-NCAM+ progenitor cell type induced pluripotent stem cell sample name E-MTAB-6018:Apex_iPSC_iN14d.fastq.gz time 14 ERS1886966 SAMEA104227948 9606 Homo sapiens Protocols: 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. Induced iPSC neurons (iPSC-iNs): The cell surface markers SSEA4+ and TRA1-81+ were used for FACS-based purification of iPSCs. For iPSC-iNs, the human cDNA sequence for Ngn2 and the sequence for eGFP were linked by a 2A sequence and cloned into the tet-inducible pLV-XTP backbone (Clonetech). IPSCs were transduced with lentiviral particles for pLV-X-UbC:TetOn (UtO) and pLV-X-Ngn2:2A:eGFP (N2AG) and were selected in the presence of puromycin (0.5 μg/ml, Sigma Aldrich) and G418 (100 μg/ml, Life technologies). For iPSC-iN conversion, iPSCs were transferred into PluriPro monolayer conditions (Cell Guidance Systems) and, 24 hrs after plating, were induced with doxycycline (2 μg/ml: Sigma Aldrich) for two days. For further maturation, iNs were switched to Neuron Maturation Media (NMM) based on DMEM:F12/Neurobasal (both Gibco) (1:1) as used for directed differentiation. NMM contains the following supplements: N2 supplement (1x, Gibco), B27 supplement (1x, Gibco), Laminin (1 μg/ml, Life Technologies) and doxycycline (2 μg/ml: Sigma Aldrich). Medium was changed every other day. Differentiating iPSC-iNs were purified by gating on eGFP+/PSA-NCAM+ neurons at 3, 7 and 14 days after conversion. 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). ENA first public 2018-01-01 ENA last update 2017-08-25 External Id SAMEA104227948 INSDC center alias Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC center name Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC first public 2018-01-01T17:01:42Z INSDC last update 2017-08-25T17:29:34Z INSDC status public Submitter Id E-MTAB-6018:Apex_iPSC_iN3d.fastq.gz broker name ArrayExpress cell type neuron common name human disease autism spectrum disorder genotype pLV-X-Ngn2-2A-eGFP (N2AG) individual Apex organism part skin phenotype Ngn2+/PSA-NCAM+ progenitor cell type induced pluripotent stem cell sample name E-MTAB-6018:Apex_iPSC_iN3d.fastq.gz time 3 ERS1886967 SAMEA104227949 9606 Homo sapiens Protocols: 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. Induced iPSC neurons (iPSC-iNs): The cell surface markers SSEA4+ and TRA1-81+ were used for FACS-based purification of iPSCs. For iPSC-iNs, the human cDNA sequence for Ngn2 and the sequence for eGFP were linked by a 2A sequence and cloned into the tet-inducible pLV-XTP backbone (Clonetech). IPSCs were transduced with lentiviral particles for pLV-X-UbC:TetOn (UtO) and pLV-X-Ngn2:2A:eGFP (N2AG) and were selected in the presence of puromycin (0.5 μg/ml, Sigma Aldrich) and G418 (100 μg/ml, Life technologies). For iPSC-iN conversion, iPSCs were transferred into PluriPro monolayer conditions (Cell Guidance Systems) and, 24 hrs after plating, were induced with doxycycline (2 μg/ml: Sigma Aldrich) for two days. For further maturation, iNs were switched to Neuron Maturation Media (NMM) based on DMEM:F12/Neurobasal (both Gibco) (1:1) as used for directed differentiation. NMM contains the following supplements: N2 supplement (1x, Gibco), B27 supplement (1x, Gibco), Laminin (1 μg/ml, Life Technologies) and doxycycline (2 μg/ml: Sigma Aldrich). Medium was changed every other day. Differentiating iPSC-iNs were purified by gating on eGFP+/PSA-NCAM+ neurons at 3, 7 and 14 days after conversion. 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). ENA first public 2018-01-01 ENA last update 2017-08-25 External Id SAMEA104227949 INSDC center alias Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC center name Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC first public 2018-01-01T17:01:42Z INSDC last update 2017-08-25T17:29:34Z INSDC status public Submitter Id E-MTAB-6018:Apex_iPSC_iN7d.fastq.gz broker name ArrayExpress cell type neuron common name human disease autism spectrum disorder genotype pLV-X-Ngn2-2A-eGFP (N2AG) individual Apex organism part skin phenotype Ngn2+/PSA-NCAM+ progenitor cell type induced pluripotent stem cell sample name E-MTAB-6018:Apex_iPSC_iN7d.fastq.gz time 7 ERS1886968 SAMEA104227950 9606 Homo sapiens Protocols: 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). ENA first public 2018-01-01 ENA last update 2017-08-25 External Id SAMEA104227950 INSDC center alias Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC center name Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC first public 2018-01-01T17:01:42Z INSDC last update 2017-08-25T17:29:34Z INSDC status public Submitter Id E-MTAB-6018:Arch_D14.fastq.gz broker name ArrayExpress cell type neuron common name human disease autism spectrum disorder genotype pRV-CAG:LckN-eGFP individual Arch organism part skin phenotype Ngn2+/PSA-NCAM+ progenitor cell type neuronal stem cell sample name E-MTAB-6018:Arch_D14.fastq.gz time 14 ERS1886969 SAMEA104227951 9606 Homo sapiens Protocols: 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). ENA first public 2018-01-01 ENA last update 2017-08-25 External Id SAMEA104227951 INSDC center alias Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC center name Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC first public 2018-01-01T17:01:42Z INSDC last update 2017-08-25T17:29:34Z INSDC status public Submitter Id E-MTAB-6018:Arch_D2.fastq.gz broker name ArrayExpress cell type neuron common name human disease autism spectrum disorder genotype pRV-CAG:LckN-eGFP individual Arch organism part skin phenotype Ngn2+/PSA-NCAM+ progenitor cell type neuronal stem cell sample name E-MTAB-6018:Arch_D2.fastq.gz time 2 ERS1886971 SAMEA104227953 9606 Homo sapiens Protocols: 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). ENA first public 2018-01-01 ENA last update 2017-08-25 External Id SAMEA104227953 INSDC center alias Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC center name Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC first public 2018-01-01T17:01:42Z INSDC last update 2017-08-25T17:29:34Z INSDC status public Submitter Id E-MTAB-6018:Arch_D7.fastq.gz broker name ArrayExpress cell type neuron common name human disease autism spectrum disorder genotype pRV-CAG:LckN-eGFP individual Arch organism part skin phenotype Ngn2+/PSA-NCAM+ progenitor cell type neuronal stem cell sample name E-MTAB-6018:Arch_D7.fastq.gz time 7 ERS1886972 SAMEA104227954 9606 Homo sapiens Protocols: 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. Induced iPSC neurons (iPSC-iNs): The cell surface markers SSEA4+ and TRA1-81+ were used for FACS-based purification of iPSCs. For iPSC-iNs, the human cDNA sequence for Ngn2 and the sequence for eGFP were linked by a 2A sequence and cloned into the tet-inducible pLV-XTP backbone (Clonetech). IPSCs were transduced with lentiviral particles for pLV-X-UbC:TetOn (UtO) and pLV-X-Ngn2:2A:eGFP (N2AG) and were selected in the presence of puromycin (0.5 μg/ml, Sigma Aldrich) and G418 (100 μg/ml, Life technologies). For iPSC-iN conversion, iPSCs were transferred into PluriPro monolayer conditions (Cell Guidance Systems) and, 24 hrs after plating, were induced with doxycycline (2 μg/ml: Sigma Aldrich) for two days. For further maturation, iNs were switched to Neuron Maturation Media (NMM) based on DMEM:F12/Neurobasal (both Gibco) (1:1) as used for directed differentiation. NMM contains the following supplements: N2 supplement (1x, Gibco), B27 supplement (1x, Gibco), Laminin (1 μg/ml, Life Technologies) and doxycycline (2 μg/ml: Sigma Aldrich). Medium was changed every other day. Differentiating iPSC-iNs were purified by gating on eGFP+/PSA-NCAM+ neurons at 3, 7 and 14 days after conversion. 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). ENA first public 2018-01-01 ENA last update 2017-08-25 External Id SAMEA104227954 INSDC center alias Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC center name Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC first public 2018-01-01T17:01:42Z INSDC last update 2017-08-25T17:29:34Z INSDC status public Submitter Id E-MTAB-6018:Arch_iPSC_iN14d.fastq.gz broker name ArrayExpress cell type neuron common name human disease autism spectrum disorder genotype pLV-X-Ngn2-2A-eGFP (N2AG) individual Arch organism part skin phenotype Ngn2+/PSA-NCAM+ progenitor cell type induced pluripotent stem cell sample name E-MTAB-6018:Arch_iPSC_iN14d.fastq.gz time 14 ERS1886973 SAMEA104227955 9606 Homo sapiens Protocols: 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. Induced iPSC neurons (iPSC-iNs): The cell surface markers SSEA4+ and TRA1-81+ were used for FACS-based purification of iPSCs. For iPSC-iNs, the human cDNA sequence for Ngn2 and the sequence for eGFP were linked by a 2A sequence and cloned into the tet-inducible pLV-XTP backbone (Clonetech). IPSCs were transduced with lentiviral particles for pLV-X-UbC:TetOn (UtO) and pLV-X-Ngn2:2A:eGFP (N2AG) and were selected in the presence of puromycin (0.5 μg/ml, Sigma Aldrich) and G418 (100 μg/ml, Life technologies). For iPSC-iN conversion, iPSCs were transferred into PluriPro monolayer conditions (Cell Guidance Systems) and, 24 hrs after plating, were induced with doxycycline (2 μg/ml: Sigma Aldrich) for two days. For further maturation, iNs were switched to Neuron Maturation Media (NMM) based on DMEM:F12/Neurobasal (both Gibco) (1:1) as used for directed differentiation. NMM contains the following supplements: N2 supplement (1x, Gibco), B27 supplement (1x, Gibco), Laminin (1 μg/ml, Life Technologies) and doxycycline (2 μg/ml: Sigma Aldrich). Medium was changed every other day. Differentiating iPSC-iNs were purified by gating on eGFP+/PSA-NCAM+ neurons at 3, 7 and 14 days after conversion. 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). ENA first public 2018-01-01 ENA last update 2017-08-25 External Id SAMEA104227955 INSDC center alias Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC center name Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC first public 2018-01-01T17:01:42Z INSDC last update 2017-08-25T17:29:34Z INSDC status public Submitter Id E-MTAB-6018:Arch_iPSC_iN3d.fastq.gz broker name ArrayExpress cell type neuron common name human disease autism spectrum disorder genotype pLV-X-Ngn2-2A-eGFP (N2AG) individual Arch organism part skin phenotype Ngn2+/PSA-NCAM+ progenitor cell type induced pluripotent stem cell sample name E-MTAB-6018:Arch_iPSC_iN3d.fastq.gz time 3 ERS1886976 SAMEA104227958 9606 Homo sapiens Protocols: 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). ENA first public 2018-01-01 ENA last update 2017-08-25 External Id SAMEA104227958 INSDC center alias Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC center name Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC first public 2018-01-01T17:01:42Z INSDC last update 2017-08-25T17:29:34Z INSDC status public Submitter Id E-MTAB-6018:Avid_D2.fastq.gz broker name ArrayExpress cell type neuron common name human disease autism spectrum disorder genotype pRV-CAG:LckN-eGFP individual Avid organism part skin phenotype Ngn2+/PSA-NCAM+ progenitor cell type neuronal stem cell sample name E-MTAB-6018:Avid_D2.fastq.gz time 2 ERS1886977 SAMEA104227959 9606 Homo sapiens Protocols: 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). ENA first public 2018-01-01 ENA last update 2017-08-25 External Id SAMEA104227959 INSDC center alias Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC center name Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC first public 2018-01-01T17:01:42Z INSDC last update 2017-08-25T17:29:34Z INSDC status public Submitter Id E-MTAB-6018:Avid_D4.fastq.gz broker name ArrayExpress cell type neuron common name human disease autism spectrum disorder genotype pRV-CAG:LckN-eGFP individual Avid organism part skin phenotype Ngn2+/PSA-NCAM+ progenitor cell type neuronal stem cell sample name E-MTAB-6018:Avid_D4.fastq.gz time 4 ERS1886980 SAMEA104227962 9606 Homo sapiens Protocols: 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. Induced iPSC neurons (iPSC-iNs): The cell surface markers SSEA4+ and TRA1-81+ were used for FACS-based purification of iPSCs. For iPSC-iNs, the human cDNA sequence for Ngn2 and the sequence for eGFP were linked by a 2A sequence and cloned into the tet-inducible pLV-XTP backbone (Clonetech). IPSCs were transduced with lentiviral particles for pLV-X-UbC:TetOn (UtO) and pLV-X-Ngn2:2A:eGFP (N2AG) and were selected in the presence of puromycin (0.5 μg/ml, Sigma Aldrich) and G418 (100 μg/ml, Life technologies). For iPSC-iN conversion, iPSCs were transferred into PluriPro monolayer conditions (Cell Guidance Systems) and, 24 hrs after plating, were induced with doxycycline (2 μg/ml: Sigma Aldrich) for two days. For further maturation, iNs were switched to Neuron Maturation Media (NMM) based on DMEM:F12/Neurobasal (both Gibco) (1:1) as used for directed differentiation. NMM contains the following supplements: N2 supplement (1x, Gibco), B27 supplement (1x, Gibco), Laminin (1 μg/ml, Life Technologies) and doxycycline (2 μg/ml: Sigma Aldrich). Medium was changed every other day. Differentiating iPSC-iNs were purified by gating on eGFP+/PSA-NCAM+ neurons at 3, 7 and 14 days after conversion. 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). ENA first public 2018-01-01 ENA last update 2017-08-25 External Id SAMEA104227962 INSDC center alias Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC center name Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC first public 2018-01-01T17:01:42Z INSDC last update 2017-08-25T17:29:34Z INSDC status public Submitter Id E-MTAB-6018:Avid_iPSC_iN3d.fastq.gz broker name ArrayExpress cell type neuron common name human disease autism spectrum disorder genotype pLV-X-Ngn2-2A-eGFP (N2AG) individual Avid organism part skin phenotype Ngn2+/PSA-NCAM+ progenitor cell type induced pluripotent stem cell sample name E-MTAB-6018:Avid_iPSC_iN3d.fastq.gz time 3 ERS1886982 SAMEA104227964 9606 Homo sapiens Protocols: 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). ENA first public 2018-01-01 ENA last update 2017-08-25 External Id SAMEA104227964 INSDC center alias Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC center name Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC first public 2018-01-01T17:01:42Z INSDC last update 2017-08-25T17:29:34Z INSDC status public Submitter Id E-MTAB-6018:Cent_D14.fastq.gz broker name ArrayExpress cell type neuron common name human disease normal genotype pRV-CAG:LckN-eGFP individual Cent organism part skin phenotype Ngn2+/PSA-NCAM+ progenitor cell type neuronal stem cell sample name E-MTAB-6018:Cent_D14.fastq.gz time 14 ERS1886983 SAMEA104227965 9606 Homo sapiens Protocols: 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). ENA first public 2018-01-01 ENA last update 2017-08-25 External Id SAMEA104227965 INSDC center alias Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC center name Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC first public 2018-01-01T17:01:42Z INSDC last update 2017-08-25T17:29:34Z INSDC status public Submitter Id E-MTAB-6018:Cent_D2.fastq.gz broker name ArrayExpress cell type neuron common name human disease normal genotype pRV-CAG:LckN-eGFP individual Cent organism part skin phenotype Ngn2+/PSA-NCAM+ progenitor cell type neuronal stem cell sample name E-MTAB-6018:Cent_D2.fastq.gz time 2 ERS1886985 SAMEA104227967 9606 Homo sapiens Protocols: 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). ENA first public 2018-01-01 ENA last update 2017-08-25 External Id SAMEA104227967 INSDC center alias Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC center name Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC first public 2018-01-01T17:01:42Z INSDC last update 2017-08-25T17:29:34Z INSDC status public Submitter Id E-MTAB-6018:Cent_D7.fastq.gz broker name ArrayExpress cell type neuron common name human disease normal genotype pRV-CAG:LckN-eGFP individual Cent organism part skin phenotype Ngn2+/PSA-NCAM+ progenitor cell type neuronal stem cell sample name E-MTAB-6018:Cent_D7.fastq.gz time 7 ERS1886987 SAMEA104227969 9606 Homo sapiens Protocols: 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. Induced iPSC neurons (iPSC-iNs): The cell surface markers SSEA4+ and TRA1-81+ were used for FACS-based purification of iPSCs. For iPSC-iNs, the human cDNA sequence for Ngn2 and the sequence for eGFP were linked by a 2A sequence and cloned into the tet-inducible pLV-XTP backbone (Clonetech). IPSCs were transduced with lentiviral particles for pLV-X-UbC:TetOn (UtO) and pLV-X-Ngn2:2A:eGFP (N2AG) and were selected in the presence of puromycin (0.5 μg/ml, Sigma Aldrich) and G418 (100 μg/ml, Life technologies). For iPSC-iN conversion, iPSCs were transferred into PluriPro monolayer conditions (Cell Guidance Systems) and, 24 hrs after plating, were induced with doxycycline (2 μg/ml: Sigma Aldrich) for two days. For further maturation, iNs were switched to Neuron Maturation Media (NMM) based on DMEM:F12/Neurobasal (both Gibco) (1:1) as used for directed differentiation. NMM contains the following supplements: N2 supplement (1x, Gibco), B27 supplement (1x, Gibco), Laminin (1 μg/ml, Life Technologies) and doxycycline (2 μg/ml: Sigma Aldrich). Medium was changed every other day. Differentiating iPSC-iNs were purified by gating on eGFP+/PSA-NCAM+ neurons at 3, 7 and 14 days after conversion. 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). ENA first public 2018-01-01 ENA last update 2017-08-25 External Id SAMEA104227969 INSDC center alias Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC center name Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC first public 2018-01-01T17:01:42Z INSDC last update 2017-08-25T17:29:35Z INSDC status public Submitter Id E-MTAB-6018:Cent_iPSC_iN3d.fastq.gz broker name ArrayExpress cell type neuron common name human disease normal genotype pLV-X-Ngn2-2A-eGFP (N2AG) individual Cent organism part skin phenotype Ngn2+/PSA-NCAM+ progenitor cell type induced pluripotent stem cell sample name E-MTAB-6018:Cent_iPSC_iN3d.fastq.gz time 3 ERS1886989 SAMEA104227971 9606 Homo sapiens Protocols: 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). ENA first public 2018-01-01 ENA last update 2017-08-25 External Id SAMEA104227971 INSDC center alias Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC center name Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC first public 2018-01-01T17:01:42Z INSDC last update 2017-08-25T17:29:35Z INSDC status public Submitter Id E-MTAB-6018:Chap_D14.fastq.gz broker name ArrayExpress cell type neuron common name human disease normal genotype pRV-CAG:LckN-eGFP individual Chap organism part skin phenotype Ngn2+/PSA-NCAM+ progenitor cell type neuronal stem cell sample name E-MTAB-6018:Chap_D14.fastq.gz time 14 ERS1886991 SAMEA104227973 9606 Homo sapiens Protocols: 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). ENA first public 2018-01-01 ENA last update 2017-08-25 External Id SAMEA104227973 INSDC center alias Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC center name Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC first public 2018-01-01T17:01:42Z INSDC last update 2017-08-25T17:29:35Z INSDC status public Submitter Id E-MTAB-6018:Chap_D4.fastq.gz broker name ArrayExpress cell type neuron common name human disease normal genotype pRV-CAG:LckN-eGFP individual Chap organism part skin phenotype Ngn2+/PSA-NCAM+ progenitor cell type neuronal stem cell sample name E-MTAB-6018:Chap_D4.fastq.gz time 4 ERS1886992 SAMEA104227974 9606 Homo sapiens Protocols: 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). ENA first public 2018-01-01 ENA last update 2017-08-25 External Id SAMEA104227974 INSDC center alias Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC center name Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC first public 2018-01-01T17:01:42Z INSDC last update 2017-08-25T17:29:35Z INSDC status public Submitter Id E-MTAB-6018:Chap_D7.fastq.gz broker name ArrayExpress cell type neuron common name human disease normal genotype pRV-CAG:LckN-eGFP individual Chap organism part skin phenotype Ngn2+/PSA-NCAM+ progenitor cell type neuronal stem cell sample name E-MTAB-6018:Chap_D7.fastq.gz time 7 ERS1886994 SAMEA104227976 9606 Homo sapiens Protocols: 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. Induced iPSC neurons (iPSC-iNs): The cell surface markers SSEA4+ and TRA1-81+ were used for FACS-based purification of iPSCs. For iPSC-iNs, the human cDNA sequence for Ngn2 and the sequence for eGFP were linked by a 2A sequence and cloned into the tet-inducible pLV-XTP backbone (Clonetech). IPSCs were transduced with lentiviral particles for pLV-X-UbC:TetOn (UtO) and pLV-X-Ngn2:2A:eGFP (N2AG) and were selected in the presence of puromycin (0.5 μg/ml, Sigma Aldrich) and G418 (100 μg/ml, Life technologies). For iPSC-iN conversion, iPSCs were transferred into PluriPro monolayer conditions (Cell Guidance Systems) and, 24 hrs after plating, were induced with doxycycline (2 μg/ml: Sigma Aldrich) for two days. For further maturation, iNs were switched to Neuron Maturation Media (NMM) based on DMEM:F12/Neurobasal (both Gibco) (1:1) as used for directed differentiation. NMM contains the following supplements: N2 supplement (1x, Gibco), B27 supplement (1x, Gibco), Laminin (1 μg/ml, Life Technologies) and doxycycline (2 μg/ml: Sigma Aldrich). Medium was changed every other day. Differentiating iPSC-iNs were purified by gating on eGFP+/PSA-NCAM+ neurons at 3, 7 and 14 days after conversion. 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). ENA first public 2018-01-01 ENA last update 2017-08-25 External Id SAMEA104227976 INSDC center alias Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC center name Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC first public 2018-01-01T17:01:42Z INSDC last update 2017-08-25T17:29:35Z INSDC status public Submitter Id E-MTAB-6018:Chap_iPSC_iN3d.fastq.gz broker name ArrayExpress cell type neuron common name human disease normal genotype pLV-X-Ngn2-2A-eGFP (N2AG) individual Chap organism part skin phenotype Ngn2+/PSA-NCAM+ progenitor cell type induced pluripotent stem cell sample name E-MTAB-6018:Chap_iPSC_iN3d.fastq.gz time 3 ERS1886995 SAMEA104227977 9606 Homo sapiens Protocols: 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. Induced iPSC neurons (iPSC-iNs): The cell surface markers SSEA4+ and TRA1-81+ were used for FACS-based purification of iPSCs. For iPSC-iNs, the human cDNA sequence for Ngn2 and the sequence for eGFP were linked by a 2A sequence and cloned into the tet-inducible pLV-XTP backbone (Clonetech). IPSCs were transduced with lentiviral particles for pLV-X-UbC:TetOn (UtO) and pLV-X-Ngn2:2A:eGFP (N2AG) and were selected in the presence of puromycin (0.5 μg/ml, Sigma Aldrich) and G418 (100 μg/ml, Life technologies). For iPSC-iN conversion, iPSCs were transferred into PluriPro monolayer conditions (Cell Guidance Systems) and, 24 hrs after plating, were induced with doxycycline (2 μg/ml: Sigma Aldrich) for two days. For further maturation, iNs were switched to Neuron Maturation Media (NMM) based on DMEM:F12/Neurobasal (both Gibco) (1:1) as used for directed differentiation. NMM contains the following supplements: N2 supplement (1x, Gibco), B27 supplement (1x, Gibco), Laminin (1 μg/ml, Life Technologies) and doxycycline (2 μg/ml: Sigma Aldrich). Medium was changed every other day. Differentiating iPSC-iNs were purified by gating on eGFP+/PSA-NCAM+ neurons at 3, 7 and 14 days after conversion. 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). ENA first public 2018-01-01 ENA last update 2017-08-25 External Id SAMEA104227977 INSDC center alias Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC center name Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC first public 2018-01-01T17:01:42Z INSDC last update 2017-08-25T17:29:35Z INSDC status public Submitter Id E-MTAB-6018:Chap_iPSC_iN7d.fastq.gz broker name ArrayExpress cell type neuron common name human disease normal genotype pLV-X-Ngn2-2A-eGFP (N2AG) individual Chap organism part skin phenotype Ngn2+/PSA-NCAM+ progenitor cell type induced pluripotent stem cell sample name E-MTAB-6018:Chap_iPSC_iN7d.fastq.gz time 7 ERS1886997 SAMEA104227979 9606 Homo sapiens Protocols: 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). ENA first public 2018-01-01 ENA last update 2017-08-25 External Id SAMEA104227979 INSDC center alias Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC center name Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC first public 2018-01-01T17:01:42Z INSDC last update 2017-08-25T17:29:35Z INSDC status public Submitter Id E-MTAB-6018:Clay_D2.fastq.gz broker name ArrayExpress cell type neuron common name human disease normal genotype pRV-CAG:LckN-eGFP individual Clay organism part skin phenotype Ngn2+/PSA-NCAM+ progenitor cell type neuronal stem cell sample name E-MTAB-6018:Clay_D2.fastq.gz time 2 ERS1886998 SAMEA104227980 9606 Homo sapiens Protocols: 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). ENA first public 2018-01-01 ENA last update 2017-08-25 External Id SAMEA104227980 INSDC center alias Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC center name Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC first public 2018-01-01T17:01:42Z INSDC last update 2017-08-25T17:29:35Z INSDC status public Submitter Id E-MTAB-6018:Clay_D4.fastq.gz broker name ArrayExpress cell type neuron common name human disease normal genotype pRV-CAG:LckN-eGFP individual Clay organism part skin phenotype Ngn2+/PSA-NCAM+ progenitor cell type neuronal stem cell sample name E-MTAB-6018:Clay_D4.fastq.gz time 4 ERS1887000 SAMEA104227982 9606 Homo sapiens Protocols: 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. Induced iPSC neurons (iPSC-iNs): The cell surface markers SSEA4+ and TRA1-81+ were used for FACS-based purification of iPSCs. For iPSC-iNs, the human cDNA sequence for Ngn2 and the sequence for eGFP were linked by a 2A sequence and cloned into the tet-inducible pLV-XTP backbone (Clonetech). IPSCs were transduced with lentiviral particles for pLV-X-UbC:TetOn (UtO) and pLV-X-Ngn2:2A:eGFP (N2AG) and were selected in the presence of puromycin (0.5 μg/ml, Sigma Aldrich) and G418 (100 μg/ml, Life technologies). For iPSC-iN conversion, iPSCs were transferred into PluriPro monolayer conditions (Cell Guidance Systems) and, 24 hrs after plating, were induced with doxycycline (2 μg/ml: Sigma Aldrich) for two days. For further maturation, iNs were switched to Neuron Maturation Media (NMM) based on DMEM:F12/Neurobasal (both Gibco) (1:1) as used for directed differentiation. NMM contains the following supplements: N2 supplement (1x, Gibco), B27 supplement (1x, Gibco), Laminin (1 μg/ml, Life Technologies) and doxycycline (2 μg/ml: Sigma Aldrich). Medium was changed every other day. Differentiating iPSC-iNs were purified by gating on eGFP+/PSA-NCAM+ neurons at 3, 7 and 14 days after conversion. 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). ENA first public 2018-01-01 ENA last update 2017-08-25 External Id SAMEA104227982 INSDC center alias Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC center name Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC first public 2018-01-01T17:01:42Z INSDC last update 2017-08-25T17:29:35Z INSDC status public Submitter Id E-MTAB-6018:Clay_iPSC_iN14d.fastq.gz broker name ArrayExpress cell type neuron common name human disease normal genotype pLV-X-Ngn2-2A-eGFP (N2AG) individual Clay organism part skin phenotype Ngn2+/PSA-NCAM+ progenitor cell type induced pluripotent stem cell sample name E-MTAB-6018:Clay_iPSC_iN14d.fastq.gz time 14 ERS1887001 SAMEA104227983 9606 Homo sapiens Protocols: 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. Induced iPSC neurons (iPSC-iNs): The cell surface markers SSEA4+ and TRA1-81+ were used for FACS-based purification of iPSCs. For iPSC-iNs, the human cDNA sequence for Ngn2 and the sequence for eGFP were linked by a 2A sequence and cloned into the tet-inducible pLV-XTP backbone (Clonetech). IPSCs were transduced with lentiviral particles for pLV-X-UbC:TetOn (UtO) and pLV-X-Ngn2:2A:eGFP (N2AG) and were selected in the presence of puromycin (0.5 μg/ml, Sigma Aldrich) and G418 (100 μg/ml, Life technologies). For iPSC-iN conversion, iPSCs were transferred into PluriPro monolayer conditions (Cell Guidance Systems) and, 24 hrs after plating, were induced with doxycycline (2 μg/ml: Sigma Aldrich) for two days. For further maturation, iNs were switched to Neuron Maturation Media (NMM) based on DMEM:F12/Neurobasal (both Gibco) (1:1) as used for directed differentiation. NMM contains the following supplements: N2 supplement (1x, Gibco), B27 supplement (1x, Gibco), Laminin (1 μg/ml, Life Technologies) and doxycycline (2 μg/ml: Sigma Aldrich). Medium was changed every other day. Differentiating iPSC-iNs were purified by gating on eGFP+/PSA-NCAM+ neurons at 3, 7 and 14 days after conversion. 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). ENA first public 2018-01-01 ENA last update 2017-08-25 External Id SAMEA104227983 INSDC center alias Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC center name Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC first public 2018-01-01T17:01:42Z INSDC last update 2017-08-25T17:29:35Z INSDC status public Submitter Id E-MTAB-6018:Clay_iPSC_iN3d.fastq.gz broker name ArrayExpress cell type neuron common name human disease normal genotype pLV-X-Ngn2-2A-eGFP (N2AG) individual Clay organism part skin phenotype Ngn2+/PSA-NCAM+ progenitor cell type induced pluripotent stem cell sample name E-MTAB-6018:Clay_iPSC_iN3d.fastq.gz time 3 ERS1887002 SAMEA104227984 9606 Homo sapiens Protocols: 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. Induced iPSC neurons (iPSC-iNs): The cell surface markers SSEA4+ and TRA1-81+ were used for FACS-based purification of iPSCs. For iPSC-iNs, the human cDNA sequence for Ngn2 and the sequence for eGFP were linked by a 2A sequence and cloned into the tet-inducible pLV-XTP backbone (Clonetech). IPSCs were transduced with lentiviral particles for pLV-X-UbC:TetOn (UtO) and pLV-X-Ngn2:2A:eGFP (N2AG) and were selected in the presence of puromycin (0.5 μg/ml, Sigma Aldrich) and G418 (100 μg/ml, Life technologies). For iPSC-iN conversion, iPSCs were transferred into PluriPro monolayer conditions (Cell Guidance Systems) and, 24 hrs after plating, were induced with doxycycline (2 μg/ml: Sigma Aldrich) for two days. For further maturation, iNs were switched to Neuron Maturation Media (NMM) based on DMEM:F12/Neurobasal (both Gibco) (1:1) as used for directed differentiation. NMM contains the following supplements: N2 supplement (1x, Gibco), B27 supplement (1x, Gibco), Laminin (1 μg/ml, Life Technologies) and doxycycline (2 μg/ml: Sigma Aldrich). Medium was changed every other day. Differentiating iPSC-iNs were purified by gating on eGFP+/PSA-NCAM+ neurons at 3, 7 and 14 days after conversion. 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). ENA first public 2018-01-01 ENA last update 2017-08-25 External Id SAMEA104227984 INSDC center alias Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC center name Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC first public 2018-01-01T17:01:42Z INSDC last update 2017-08-25T17:29:35Z INSDC status public Submitter Id E-MTAB-6018:Clay_iPSC_iN7d.fastq.gz broker name ArrayExpress cell type neuron common name human disease normal genotype pLV-X-Ngn2-2A-eGFP (N2AG) individual Clay organism part skin phenotype Ngn2+/PSA-NCAM+ progenitor cell type induced pluripotent stem cell sample name E-MTAB-6018:Clay_iPSC_iN7d.fastq.gz time 7 ERS1887005 SAMEA104227987 9606 Homo sapiens Protocols: 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). ENA first public 2018-01-01 ENA last update 2017-08-25 External Id SAMEA104227987 INSDC center alias Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC center name Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC first public 2018-01-01T17:01:42Z INSDC last update 2017-08-25T17:29:35Z INSDC status public Submitter Id E-MTAB-6018:Clue_D4.fastq.gz broker name ArrayExpress cell type neuron common name human disease normal genotype pRV-CAG:LckN-eGFP individual Clue organism part skin phenotype Ngn2+/PSA-NCAM+ progenitor cell type neuronal stem cell sample name E-MTAB-6018:Clue_D4.fastq.gz time 4 ERS1887006 SAMEA104227988 9606 Homo sapiens Protocols: 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). ENA first public 2018-01-01 ENA last update 2017-08-25 External Id SAMEA104227988 INSDC center alias Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC center name Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC first public 2018-01-01T17:01:42Z INSDC last update 2017-08-25T17:29:35Z INSDC status public Submitter Id E-MTAB-6018:Clue_D7.fastq.gz broker name ArrayExpress cell type neuron common name human disease normal genotype pRV-CAG:LckN-eGFP individual Clue organism part skin phenotype Ngn2+/PSA-NCAM+ progenitor cell type neuronal stem cell sample name E-MTAB-6018:Clue_D7.fastq.gz time 7 ERS1887007 SAMEA104227989 9606 Homo sapiens Protocols: 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. Induced iPSC neurons (iPSC-iNs): The cell surface markers SSEA4+ and TRA1-81+ were used for FACS-based purification of iPSCs. For iPSC-iNs, the human cDNA sequence for Ngn2 and the sequence for eGFP were linked by a 2A sequence and cloned into the tet-inducible pLV-XTP backbone (Clonetech). IPSCs were transduced with lentiviral particles for pLV-X-UbC:TetOn (UtO) and pLV-X-Ngn2:2A:eGFP (N2AG) and were selected in the presence of puromycin (0.5 μg/ml, Sigma Aldrich) and G418 (100 μg/ml, Life technologies). For iPSC-iN conversion, iPSCs were transferred into PluriPro monolayer conditions (Cell Guidance Systems) and, 24 hrs after plating, were induced with doxycycline (2 μg/ml: Sigma Aldrich) for two days. For further maturation, iNs were switched to Neuron Maturation Media (NMM) based on DMEM:F12/Neurobasal (both Gibco) (1:1) as used for directed differentiation. NMM contains the following supplements: N2 supplement (1x, Gibco), B27 supplement (1x, Gibco), Laminin (1 μg/ml, Life Technologies) and doxycycline (2 μg/ml: Sigma Aldrich). Medium was changed every other day. Differentiating iPSC-iNs were purified by gating on eGFP+/PSA-NCAM+ neurons at 3, 7 and 14 days after conversion. 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). ENA first public 2018-01-01 ENA last update 2017-08-25 External Id SAMEA104227989 INSDC center alias Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC center name Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC first public 2018-01-01T17:01:42Z INSDC last update 2017-08-25T17:29:35Z INSDC status public Submitter Id E-MTAB-6018:Clue_iPSC_iN14d.fastq.gz broker name ArrayExpress cell type neuron common name human disease normal genotype pLV-X-Ngn2-2A-eGFP (N2AG) individual Clue organism part skin phenotype Ngn2+/PSA-NCAM+ progenitor cell type induced pluripotent stem cell sample name E-MTAB-6018:Clue_iPSC_iN14d.fastq.gz time 14 ERS1887009 SAMEA104227991 9606 Homo sapiens Protocols: 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. Induced iPSC neurons (iPSC-iNs): The cell surface markers SSEA4+ and TRA1-81+ were used for FACS-based purification of iPSCs. For iPSC-iNs, the human cDNA sequence for Ngn2 and the sequence for eGFP were linked by a 2A sequence and cloned into the tet-inducible pLV-XTP backbone (Clonetech). IPSCs were transduced with lentiviral particles for pLV-X-UbC:TetOn (UtO) and pLV-X-Ngn2:2A:eGFP (N2AG) and were selected in the presence of puromycin (0.5 μg/ml, Sigma Aldrich) and G418 (100 μg/ml, Life technologies). For iPSC-iN conversion, iPSCs were transferred into PluriPro monolayer conditions (Cell Guidance Systems) and, 24 hrs after plating, were induced with doxycycline (2 μg/ml: Sigma Aldrich) for two days. For further maturation, iNs were switched to Neuron Maturation Media (NMM) based on DMEM:F12/Neurobasal (both Gibco) (1:1) as used for directed differentiation. NMM contains the following supplements: N2 supplement (1x, Gibco), B27 supplement (1x, Gibco), Laminin (1 μg/ml, Life Technologies) and doxycycline (2 μg/ml: Sigma Aldrich). Medium was changed every other day. Differentiating iPSC-iNs were purified by gating on eGFP+/PSA-NCAM+ neurons at 3, 7 and 14 days after conversion. 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). ENA first public 2018-01-01 ENA last update 2017-08-25 External Id SAMEA104227991 INSDC center alias Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC center name Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC first public 2018-01-01T17:01:42Z INSDC last update 2017-08-25T17:29:35Z INSDC status public Submitter Id E-MTAB-6018:Clue_iPSC_iN7d.fastq.gz broker name ArrayExpress cell type neuron common name human disease normal genotype pLV-X-Ngn2-2A-eGFP (N2AG) individual Clue organism part skin phenotype Ngn2+/PSA-NCAM+ progenitor cell type induced pluripotent stem cell sample name E-MTAB-6018:Clue_iPSC_iN7d.fastq.gz time 7 ERS1887010 SAMEA104227992 9606 Homo sapiens Protocols: 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). ENA first public 2018-01-01 ENA last update 2017-08-25 External Id SAMEA104227992 INSDC center alias Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC center name Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC first public 2018-01-01T17:01:42Z INSDC last update 2017-08-25T17:29:35Z INSDC status public Submitter Id E-MTAB-6018:Cove_D14.fastq.gz broker name ArrayExpress cell type neuron common name human disease normal genotype pRV-CAG:LckN-eGFP individual Cove organism part skin phenotype Ngn2+/PSA-NCAM+ progenitor cell type neuronal stem cell sample name E-MTAB-6018:Cove_D14.fastq.gz time 14 ERS1887012 SAMEA104227994 9606 Homo sapiens Protocols: 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). ENA first public 2018-01-01 ENA last update 2017-08-25 External Id SAMEA104227994 INSDC center alias Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC center name Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC first public 2018-01-01T17:01:42Z INSDC last update 2017-08-25T17:29:35Z INSDC status public Submitter Id E-MTAB-6018:Cove_D4.fastq.gz broker name ArrayExpress cell type neuron common name human disease normal genotype pRV-CAG:LckN-eGFP individual Cove organism part skin phenotype Ngn2+/PSA-NCAM+ progenitor cell type neuronal stem cell sample name E-MTAB-6018:Cove_D4.fastq.gz time 4 ERS1887017 SAMEA104227999 9606 Homo sapiens Protocols: 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. Induced iPSC neurons (iPSC-iNs): The cell surface markers SSEA4+ and TRA1-81+ were used for FACS-based purification of iPSCs. For iPSC-iNs, the human cDNA sequence for Ngn2 and the sequence for eGFP were linked by a 2A sequence and cloned into the tet-inducible pLV-XTP backbone (Clonetech). IPSCs were transduced with lentiviral particles for pLV-X-UbC:TetOn (UtO) and pLV-X-Ngn2:2A:eGFP (N2AG) and were selected in the presence of puromycin (0.5 μg/ml, Sigma Aldrich) and G418 (100 μg/ml, Life technologies). For iPSC-iN conversion, iPSCs were transferred into PluriPro monolayer conditions (Cell Guidance Systems) and, 24 hrs after plating, were induced with doxycycline (2 μg/ml: Sigma Aldrich) for two days. For further maturation, iNs were switched to Neuron Maturation Media (NMM) based on DMEM:F12/Neurobasal (both Gibco) (1:1) as used for directed differentiation. NMM contains the following supplements: N2 supplement (1x, Gibco), B27 supplement (1x, Gibco), Laminin (1 μg/ml, Life Technologies) and doxycycline (2 μg/ml: Sigma Aldrich). Medium was changed every other day. Differentiating iPSC-iNs were purified by gating on eGFP+/PSA-NCAM+ neurons at 3, 7 and 14 days after conversion. 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). ENA first public 2018-01-01 ENA last update 2017-08-25 External Id SAMEA104227999 INSDC center alias Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC center name Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC first public 2018-01-01T17:01:42Z INSDC last update 2017-08-25T17:29:35Z INSDC status public Submitter Id E-MTAB-6018:Able_iPSC.fastq.gz broker name ArrayExpress cell type induced pluripotent stem cell common name human disease autism spectrum disorder genotype pLV-X-Ngn2-2A-eGFP (N2AG) individual Able organism part skin phenotype SSEA-4+/TRA1-81+ progenitor cell type fibroblast of dermis sample name E-MTAB-6018:Able_iPSC.fastq.gz ERS1887019 SAMEA104228001 9606 Homo sapiens Protocols: 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. Induced iPSC neurons (iPSC-iNs): The cell surface markers SSEA4+ and TRA1-81+ were used for FACS-based purification of iPSCs. For iPSC-iNs, the human cDNA sequence for Ngn2 and the sequence for eGFP were linked by a 2A sequence and cloned into the tet-inducible pLV-XTP backbone (Clonetech). IPSCs were transduced with lentiviral particles for pLV-X-UbC:TetOn (UtO) and pLV-X-Ngn2:2A:eGFP (N2AG) and were selected in the presence of puromycin (0.5 μg/ml, Sigma Aldrich) and G418 (100 μg/ml, Life technologies). For iPSC-iN conversion, iPSCs were transferred into PluriPro monolayer conditions (Cell Guidance Systems) and, 24 hrs after plating, were induced with doxycycline (2 μg/ml: Sigma Aldrich) for two days. For further maturation, iNs were switched to Neuron Maturation Media (NMM) based on DMEM:F12/Neurobasal (both Gibco) (1:1) as used for directed differentiation. NMM contains the following supplements: N2 supplement (1x, Gibco), B27 supplement (1x, Gibco), Laminin (1 μg/ml, Life Technologies) and doxycycline (2 μg/ml: Sigma Aldrich). Medium was changed every other day. Differentiating iPSC-iNs were purified by gating on eGFP+/PSA-NCAM+ neurons at 3, 7 and 14 days after conversion. 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). ENA first public 2018-01-01 ENA last update 2017-08-25 External Id SAMEA104228001 INSDC center alias Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC center name Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC first public 2018-01-01T17:01:42Z INSDC last update 2017-08-25T17:29:35Z INSDC status public Submitter Id E-MTAB-6018:Aero_iPSC.fastq.gz broker name ArrayExpress cell type induced pluripotent stem cell common name human disease autism spectrum disorder genotype pLV-X-Ngn2-2A-eGFP (N2AG) individual Aero organism part skin phenotype SSEA-4+/TRA1-81+ progenitor cell type fibroblast of dermis sample name E-MTAB-6018:Aero_iPSC.fastq.gz ERS1887021 SAMEA104228003 9606 Homo sapiens Protocols: 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. Induced iPSC neurons (iPSC-iNs): The cell surface markers SSEA4+ and TRA1-81+ were used for FACS-based purification of iPSCs. For iPSC-iNs, the human cDNA sequence for Ngn2 and the sequence for eGFP were linked by a 2A sequence and cloned into the tet-inducible pLV-XTP backbone (Clonetech). IPSCs were transduced with lentiviral particles for pLV-X-UbC:TetOn (UtO) and pLV-X-Ngn2:2A:eGFP (N2AG) and were selected in the presence of puromycin (0.5 μg/ml, Sigma Aldrich) and G418 (100 μg/ml, Life technologies). For iPSC-iN conversion, iPSCs were transferred into PluriPro monolayer conditions (Cell Guidance Systems) and, 24 hrs after plating, were induced with doxycycline (2 μg/ml: Sigma Aldrich) for two days. For further maturation, iNs were switched to Neuron Maturation Media (NMM) based on DMEM:F12/Neurobasal (both Gibco) (1:1) as used for directed differentiation. NMM contains the following supplements: N2 supplement (1x, Gibco), B27 supplement (1x, Gibco), Laminin (1 μg/ml, Life Technologies) and doxycycline (2 μg/ml: Sigma Aldrich). Medium was changed every other day. Differentiating iPSC-iNs were purified by gating on eGFP+/PSA-NCAM+ neurons at 3, 7 and 14 days after conversion. 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). ENA first public 2018-01-01 ENA last update 2017-08-25 External Id SAMEA104228003 INSDC center alias Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC center name Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC first public 2018-01-01T17:01:42Z INSDC last update 2017-08-25T17:29:35Z INSDC status public Submitter Id E-MTAB-6018:Ahoy_iPSC.fastq.gz broker name ArrayExpress cell type induced pluripotent stem cell common name human disease autism spectrum disorder genotype pLV-X-Ngn2-2A-eGFP (N2AG) individual Ahoy organism part skin phenotype SSEA-4+/TRA1-81+ progenitor cell type fibroblast of dermis sample name E-MTAB-6018:Ahoy_iPSC.fastq.gz ERS1886930 SAMEA104227912 9606 Homo sapiens Protocols: 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. Induced iPSC neurons (iPSC-iNs): The cell surface markers SSEA4+ and TRA1-81+ were used for FACS-based purification of iPSCs. For iPSC-iNs, the human cDNA sequence for Ngn2 and the sequence for eGFP were linked by a 2A sequence and cloned into the tet-inducible pLV-XTP backbone (Clonetech). IPSCs were transduced with lentiviral particles for pLV-X-UbC:TetOn (UtO) and pLV-X-Ngn2:2A:eGFP (N2AG) and were selected in the presence of puromycin (0.5 μg/ml, Sigma Aldrich) and G418 (100 μg/ml, Life technologies). For iPSC-iN conversion, iPSCs were transferred into PluriPro monolayer conditions (Cell Guidance Systems) and, 24 hrs after plating, were induced with doxycycline (2 μg/ml: Sigma Aldrich) for two days. For further maturation, iNs were switched to Neuron Maturation Media (NMM) based on DMEM:F12/Neurobasal (both Gibco) (1:1) as used for directed differentiation. NMM contains the following supplements: N2 supplement (1x, Gibco), B27 supplement (1x, Gibco), Laminin (1 μg/ml, Life Technologies) and doxycycline (2 μg/ml: Sigma Aldrich). Medium was changed every other day. Differentiating iPSC-iNs were purified by gating on eGFP+/PSA-NCAM+ neurons at 3, 7 and 14 days after conversion. 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). ENA first public 2018-01-01 ENA last update 2017-08-25 External Id SAMEA104227912 INSDC center alias Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC center name Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC first public 2018-01-01T17:01:42Z INSDC last update 2017-08-25T17:29:33Z INSDC status public Submitter Id E-MTAB-6018:Able_iPSC_iN14d.fastq.gz broker name ArrayExpress cell type neuron common name human disease autism spectrum disorder genotype pLV-X-Ngn2-2A-eGFP (N2AG) individual Able organism part skin phenotype Ngn2+/PSA-NCAM+ progenitor cell type induced pluripotent stem cell sample name E-MTAB-6018:Able_iPSC_iN14d.fastq.gz time 14 ERS1886970 SAMEA104227952 9606 Homo sapiens Protocols: 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). ENA first public 2018-01-01 ENA last update 2017-08-25 External Id SAMEA104227952 INSDC center alias Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC center name Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC first public 2018-01-01T17:01:42Z INSDC last update 2017-08-25T17:29:34Z INSDC status public Submitter Id E-MTAB-6018:Arch_D4.fastq.gz broker name ArrayExpress cell type neuron common name human disease autism spectrum disorder genotype pRV-CAG:LckN-eGFP individual Arch organism part skin phenotype Ngn2+/PSA-NCAM+ progenitor cell type neuronal stem cell sample name E-MTAB-6018:Arch_D4.fastq.gz time 4 ERS1886974 SAMEA104227956 9606 Homo sapiens Protocols: 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. Induced iPSC neurons (iPSC-iNs): The cell surface markers SSEA4+ and TRA1-81+ were used for FACS-based purification of iPSCs. For iPSC-iNs, the human cDNA sequence for Ngn2 and the sequence for eGFP were linked by a 2A sequence and cloned into the tet-inducible pLV-XTP backbone (Clonetech). IPSCs were transduced with lentiviral particles for pLV-X-UbC:TetOn (UtO) and pLV-X-Ngn2:2A:eGFP (N2AG) and were selected in the presence of puromycin (0.5 μg/ml, Sigma Aldrich) and G418 (100 μg/ml, Life technologies). For iPSC-iN conversion, iPSCs were transferred into PluriPro monolayer conditions (Cell Guidance Systems) and, 24 hrs after plating, were induced with doxycycline (2 μg/ml: Sigma Aldrich) for two days. For further maturation, iNs were switched to Neuron Maturation Media (NMM) based on DMEM:F12/Neurobasal (both Gibco) (1:1) as used for directed differentiation. NMM contains the following supplements: N2 supplement (1x, Gibco), B27 supplement (1x, Gibco), Laminin (1 μg/ml, Life Technologies) and doxycycline (2 μg/ml: Sigma Aldrich). Medium was changed every other day. Differentiating iPSC-iNs were purified by gating on eGFP+/PSA-NCAM+ neurons at 3, 7 and 14 days after conversion. 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). ENA first public 2018-01-01 ENA last update 2017-08-25 External Id SAMEA104227956 INSDC center alias Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC center name Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC first public 2018-01-01T17:01:42Z INSDC last update 2017-08-25T17:29:34Z INSDC status public Submitter Id E-MTAB-6018:Arch_iPSC_iN7d.fastq.gz broker name ArrayExpress cell type neuron common name human disease autism spectrum disorder genotype pLV-X-Ngn2-2A-eGFP (N2AG) individual Arch organism part skin phenotype Ngn2+/PSA-NCAM+ progenitor cell type induced pluripotent stem cell sample name E-MTAB-6018:Arch_iPSC_iN7d.fastq.gz time 7 ERS1886975 SAMEA104227957 9606 Homo sapiens Protocols: 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). ENA first public 2018-01-01 ENA last update 2017-08-25 External Id SAMEA104227957 INSDC center alias Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC center name Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC first public 2018-01-01T17:01:42Z INSDC last update 2017-08-25T17:29:34Z INSDC status public Submitter Id E-MTAB-6018:Avid_D14.fastq.gz broker name ArrayExpress cell type neuron common name human disease autism spectrum disorder genotype pRV-CAG:LckN-eGFP individual Avid organism part skin phenotype Ngn2+/PSA-NCAM+ progenitor cell type neuronal stem cell sample name E-MTAB-6018:Avid_D14.fastq.gz time 14 ERS1886978 SAMEA104227960 9606 Homo sapiens Protocols: 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). ENA first public 2018-01-01 ENA last update 2017-08-25 External Id SAMEA104227960 INSDC center alias Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC center name Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC first public 2018-01-01T17:01:42Z INSDC last update 2017-08-25T17:29:34Z INSDC status public Submitter Id E-MTAB-6018:Avid_D7.fastq.gz broker name ArrayExpress cell type neuron common name human disease autism spectrum disorder genotype pRV-CAG:LckN-eGFP individual Avid organism part skin phenotype Ngn2+/PSA-NCAM+ progenitor cell type neuronal stem cell sample name E-MTAB-6018:Avid_D7.fastq.gz time 7 ERS1886979 SAMEA104227961 9606 Homo sapiens Protocols: 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. Induced iPSC neurons (iPSC-iNs): The cell surface markers SSEA4+ and TRA1-81+ were used for FACS-based purification of iPSCs. For iPSC-iNs, the human cDNA sequence for Ngn2 and the sequence for eGFP were linked by a 2A sequence and cloned into the tet-inducible pLV-XTP backbone (Clonetech). IPSCs were transduced with lentiviral particles for pLV-X-UbC:TetOn (UtO) and pLV-X-Ngn2:2A:eGFP (N2AG) and were selected in the presence of puromycin (0.5 μg/ml, Sigma Aldrich) and G418 (100 μg/ml, Life technologies). For iPSC-iN conversion, iPSCs were transferred into PluriPro monolayer conditions (Cell Guidance Systems) and, 24 hrs after plating, were induced with doxycycline (2 μg/ml: Sigma Aldrich) for two days. For further maturation, iNs were switched to Neuron Maturation Media (NMM) based on DMEM:F12/Neurobasal (both Gibco) (1:1) as used for directed differentiation. NMM contains the following supplements: N2 supplement (1x, Gibco), B27 supplement (1x, Gibco), Laminin (1 μg/ml, Life Technologies) and doxycycline (2 μg/ml: Sigma Aldrich). Medium was changed every other day. Differentiating iPSC-iNs were purified by gating on eGFP+/PSA-NCAM+ neurons at 3, 7 and 14 days after conversion. 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). ENA first public 2018-01-01 ENA last update 2017-08-25 External Id SAMEA104227961 INSDC center alias Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC center name Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC first public 2018-01-01T17:01:42Z INSDC last update 2017-08-25T17:29:34Z INSDC status public Submitter Id E-MTAB-6018:Avid_iPSC_iN14d.fastq.gz broker name ArrayExpress cell type neuron common name human disease autism spectrum disorder genotype pLV-X-Ngn2-2A-eGFP (N2AG) individual Avid organism part skin phenotype Ngn2+/PSA-NCAM+ progenitor cell type induced pluripotent stem cell sample name E-MTAB-6018:Avid_iPSC_iN14d.fastq.gz time 14 ERS1886981 SAMEA104227963 9606 Homo sapiens Protocols: 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. Induced iPSC neurons (iPSC-iNs): The cell surface markers SSEA4+ and TRA1-81+ were used for FACS-based purification of iPSCs. For iPSC-iNs, the human cDNA sequence for Ngn2 and the sequence for eGFP were linked by a 2A sequence and cloned into the tet-inducible pLV-XTP backbone (Clonetech). IPSCs were transduced with lentiviral particles for pLV-X-UbC:TetOn (UtO) and pLV-X-Ngn2:2A:eGFP (N2AG) and were selected in the presence of puromycin (0.5 μg/ml, Sigma Aldrich) and G418 (100 μg/ml, Life technologies). For iPSC-iN conversion, iPSCs were transferred into PluriPro monolayer conditions (Cell Guidance Systems) and, 24 hrs after plating, were induced with doxycycline (2 μg/ml: Sigma Aldrich) for two days. For further maturation, iNs were switched to Neuron Maturation Media (NMM) based on DMEM:F12/Neurobasal (both Gibco) (1:1) as used for directed differentiation. NMM contains the following supplements: N2 supplement (1x, Gibco), B27 supplement (1x, Gibco), Laminin (1 μg/ml, Life Technologies) and doxycycline (2 μg/ml: Sigma Aldrich). Medium was changed every other day. Differentiating iPSC-iNs were purified by gating on eGFP+/PSA-NCAM+ neurons at 3, 7 and 14 days after conversion. 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). ENA first public 2018-01-01 ENA last update 2017-08-25 External Id SAMEA104227963 INSDC center alias Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC center name Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC first public 2018-01-01T17:01:42Z INSDC last update 2017-08-25T17:29:34Z INSDC status public Submitter Id E-MTAB-6018:Avid_iPSC_iN7d.fastq.gz broker name ArrayExpress cell type neuron common name human disease autism spectrum disorder genotype pLV-X-Ngn2-2A-eGFP (N2AG) individual Avid organism part skin phenotype Ngn2+/PSA-NCAM+ progenitor cell type induced pluripotent stem cell sample name E-MTAB-6018:Avid_iPSC_iN7d.fastq.gz time 7 ERS1886984 SAMEA104227966 9606 Homo sapiens Protocols: 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). ENA first public 2018-01-01 ENA last update 2017-08-25 External Id SAMEA104227966 INSDC center alias Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC center name Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC first public 2018-01-01T17:01:42Z INSDC last update 2017-08-25T17:29:34Z INSDC status public Submitter Id E-MTAB-6018:Cent_D4.fastq.gz broker name ArrayExpress cell type neuron common name human disease normal genotype pRV-CAG:LckN-eGFP individual Cent organism part skin phenotype Ngn2+/PSA-NCAM+ progenitor cell type neuronal stem cell sample name E-MTAB-6018:Cent_D4.fastq.gz time 4 ERS1886986 SAMEA104227968 9606 Homo sapiens Protocols: 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. Induced iPSC neurons (iPSC-iNs): The cell surface markers SSEA4+ and TRA1-81+ were used for FACS-based purification of iPSCs. For iPSC-iNs, the human cDNA sequence for Ngn2 and the sequence for eGFP were linked by a 2A sequence and cloned into the tet-inducible pLV-XTP backbone (Clonetech). IPSCs were transduced with lentiviral particles for pLV-X-UbC:TetOn (UtO) and pLV-X-Ngn2:2A:eGFP (N2AG) and were selected in the presence of puromycin (0.5 μg/ml, Sigma Aldrich) and G418 (100 μg/ml, Life technologies). For iPSC-iN conversion, iPSCs were transferred into PluriPro monolayer conditions (Cell Guidance Systems) and, 24 hrs after plating, were induced with doxycycline (2 μg/ml: Sigma Aldrich) for two days. For further maturation, iNs were switched to Neuron Maturation Media (NMM) based on DMEM:F12/Neurobasal (both Gibco) (1:1) as used for directed differentiation. NMM contains the following supplements: N2 supplement (1x, Gibco), B27 supplement (1x, Gibco), Laminin (1 μg/ml, Life Technologies) and doxycycline (2 μg/ml: Sigma Aldrich). Medium was changed every other day. Differentiating iPSC-iNs were purified by gating on eGFP+/PSA-NCAM+ neurons at 3, 7 and 14 days after conversion. 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). ENA first public 2018-01-01 ENA last update 2017-08-25 External Id SAMEA104227968 INSDC center alias Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC center name Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC first public 2018-01-01T17:01:42Z INSDC last update 2017-08-25T17:29:34Z INSDC status public Submitter Id E-MTAB-6018:Cent_iPSC_iN14d.fastq.gz broker name ArrayExpress cell type neuron common name human disease normal genotype pLV-X-Ngn2-2A-eGFP (N2AG) individual Cent organism part skin phenotype Ngn2+/PSA-NCAM+ progenitor cell type induced pluripotent stem cell sample name E-MTAB-6018:Cent_iPSC_iN14d.fastq.gz time 14 ERS1886988 SAMEA104227970 9606 Homo sapiens Protocols: 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. Induced iPSC neurons (iPSC-iNs): The cell surface markers SSEA4+ and TRA1-81+ were used for FACS-based purification of iPSCs. For iPSC-iNs, the human cDNA sequence for Ngn2 and the sequence for eGFP were linked by a 2A sequence and cloned into the tet-inducible pLV-XTP backbone (Clonetech). IPSCs were transduced with lentiviral particles for pLV-X-UbC:TetOn (UtO) and pLV-X-Ngn2:2A:eGFP (N2AG) and were selected in the presence of puromycin (0.5 μg/ml, Sigma Aldrich) and G418 (100 μg/ml, Life technologies). For iPSC-iN conversion, iPSCs were transferred into PluriPro monolayer conditions (Cell Guidance Systems) and, 24 hrs after plating, were induced with doxycycline (2 μg/ml: Sigma Aldrich) for two days. For further maturation, iNs were switched to Neuron Maturation Media (NMM) based on DMEM:F12/Neurobasal (both Gibco) (1:1) as used for directed differentiation. NMM contains the following supplements: N2 supplement (1x, Gibco), B27 supplement (1x, Gibco), Laminin (1 μg/ml, Life Technologies) and doxycycline (2 μg/ml: Sigma Aldrich). Medium was changed every other day. Differentiating iPSC-iNs were purified by gating on eGFP+/PSA-NCAM+ neurons at 3, 7 and 14 days after conversion. 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). ENA first public 2018-01-01 ENA last update 2017-08-25 External Id SAMEA104227970 INSDC center alias Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC center name Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC first public 2018-01-01T17:01:42Z INSDC last update 2017-08-25T17:29:35Z INSDC status public Submitter Id E-MTAB-6018:Cent_iPSC_iN7d.fastq.gz broker name ArrayExpress cell type neuron common name human disease normal genotype pLV-X-Ngn2-2A-eGFP (N2AG) individual Cent organism part skin phenotype Ngn2+/PSA-NCAM+ progenitor cell type induced pluripotent stem cell sample name E-MTAB-6018:Cent_iPSC_iN7d.fastq.gz time 7 ERS1886990 SAMEA104227972 9606 Homo sapiens Protocols: 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). ENA first public 2018-01-01 ENA last update 2017-08-25 External Id SAMEA104227972 INSDC center alias Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC center name Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC first public 2018-01-01T17:01:42Z INSDC last update 2017-08-25T17:29:35Z INSDC status public Submitter Id E-MTAB-6018:Chap_D2.fastq.gz broker name ArrayExpress cell type neuron common name human disease normal genotype pRV-CAG:LckN-eGFP individual Chap organism part skin phenotype Ngn2+/PSA-NCAM+ progenitor cell type neuronal stem cell sample name E-MTAB-6018:Chap_D2.fastq.gz time 2 ERS1886993 SAMEA104227975 9606 Homo sapiens Protocols: 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. Induced iPSC neurons (iPSC-iNs): The cell surface markers SSEA4+ and TRA1-81+ were used for FACS-based purification of iPSCs. For iPSC-iNs, the human cDNA sequence for Ngn2 and the sequence for eGFP were linked by a 2A sequence and cloned into the tet-inducible pLV-XTP backbone (Clonetech). IPSCs were transduced with lentiviral particles for pLV-X-UbC:TetOn (UtO) and pLV-X-Ngn2:2A:eGFP (N2AG) and were selected in the presence of puromycin (0.5 μg/ml, Sigma Aldrich) and G418 (100 μg/ml, Life technologies). For iPSC-iN conversion, iPSCs were transferred into PluriPro monolayer conditions (Cell Guidance Systems) and, 24 hrs after plating, were induced with doxycycline (2 μg/ml: Sigma Aldrich) for two days. For further maturation, iNs were switched to Neuron Maturation Media (NMM) based on DMEM:F12/Neurobasal (both Gibco) (1:1) as used for directed differentiation. NMM contains the following supplements: N2 supplement (1x, Gibco), B27 supplement (1x, Gibco), Laminin (1 μg/ml, Life Technologies) and doxycycline (2 μg/ml: Sigma Aldrich). Medium was changed every other day. Differentiating iPSC-iNs were purified by gating on eGFP+/PSA-NCAM+ neurons at 3, 7 and 14 days after conversion. 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). ENA first public 2018-01-01 ENA last update 2017-08-25 External Id SAMEA104227975 INSDC center alias Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC center name Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC first public 2018-01-01T17:01:42Z INSDC last update 2017-08-25T17:29:35Z INSDC status public Submitter Id E-MTAB-6018:Chap_iPSC_iN14d.fastq.gz broker name ArrayExpress cell type neuron common name human disease normal genotype pLV-X-Ngn2-2A-eGFP (N2AG) individual Chap organism part skin phenotype Ngn2+/PSA-NCAM+ progenitor cell type induced pluripotent stem cell sample name E-MTAB-6018:Chap_iPSC_iN14d.fastq.gz time 14 ERS1886996 SAMEA104227978 9606 Homo sapiens Protocols: 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). ENA first public 2018-01-01 ENA last update 2017-08-25 External Id SAMEA104227978 INSDC center alias Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC center name Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC first public 2018-01-01T17:01:42Z INSDC last update 2017-08-25T17:29:35Z INSDC status public Submitter Id E-MTAB-6018:Clay_D14.fastq.gz broker name ArrayExpress cell type neuron common name human disease normal genotype pRV-CAG:LckN-eGFP individual Clay organism part skin phenotype Ngn2+/PSA-NCAM+ progenitor cell type neuronal stem cell sample name E-MTAB-6018:Clay_D14.fastq.gz time 14 ERS1886999 SAMEA104227981 9606 Homo sapiens Protocols: 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). ENA first public 2018-01-01 ENA last update 2017-08-25 External Id SAMEA104227981 INSDC center alias Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC center name Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC first public 2018-01-01T17:01:42Z INSDC last update 2017-08-25T17:29:35Z INSDC status public Submitter Id E-MTAB-6018:Clay_D7.fastq.gz broker name ArrayExpress cell type neuron common name human disease normal genotype pRV-CAG:LckN-eGFP individual Clay organism part skin phenotype Ngn2+/PSA-NCAM+ progenitor cell type neuronal stem cell sample name E-MTAB-6018:Clay_D7.fastq.gz time 7 ERS1887003 SAMEA104227985 9606 Homo sapiens Protocols: 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). ENA first public 2018-01-01 ENA last update 2017-08-25 External Id SAMEA104227985 INSDC center alias Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC center name Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC first public 2018-01-01T17:01:42Z INSDC last update 2017-08-25T17:29:35Z INSDC status public Submitter Id E-MTAB-6018:Clue_D14.fastq.gz broker name ArrayExpress cell type neuron common name human disease normal genotype pRV-CAG:LckN-eGFP individual Clue organism part skin phenotype Ngn2+/PSA-NCAM+ progenitor cell type neuronal stem cell sample name E-MTAB-6018:Clue_D14.fastq.gz time 14 ERS1887004 SAMEA104227986 9606 Homo sapiens Protocols: 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). ENA first public 2018-01-01 ENA last update 2017-08-25 External Id SAMEA104227986 INSDC center alias Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC center name Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC first public 2018-01-01T17:01:42Z INSDC last update 2017-08-25T17:29:35Z INSDC status public Submitter Id E-MTAB-6018:Clue_D2.fastq.gz broker name ArrayExpress cell type neuron common name human disease normal genotype pRV-CAG:LckN-eGFP individual Clue organism part skin phenotype Ngn2+/PSA-NCAM+ progenitor cell type neuronal stem cell sample name E-MTAB-6018:Clue_D2.fastq.gz time 2 ERS1887008 SAMEA104227990 9606 Homo sapiens Protocols: 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. Induced iPSC neurons (iPSC-iNs): The cell surface markers SSEA4+ and TRA1-81+ were used for FACS-based purification of iPSCs. For iPSC-iNs, the human cDNA sequence for Ngn2 and the sequence for eGFP were linked by a 2A sequence and cloned into the tet-inducible pLV-XTP backbone (Clonetech). IPSCs were transduced with lentiviral particles for pLV-X-UbC:TetOn (UtO) and pLV-X-Ngn2:2A:eGFP (N2AG) and were selected in the presence of puromycin (0.5 μg/ml, Sigma Aldrich) and G418 (100 μg/ml, Life technologies). For iPSC-iN conversion, iPSCs were transferred into PluriPro monolayer conditions (Cell Guidance Systems) and, 24 hrs after plating, were induced with doxycycline (2 μg/ml: Sigma Aldrich) for two days. For further maturation, iNs were switched to Neuron Maturation Media (NMM) based on DMEM:F12/Neurobasal (both Gibco) (1:1) as used for directed differentiation. NMM contains the following supplements: N2 supplement (1x, Gibco), B27 supplement (1x, Gibco), Laminin (1 μg/ml, Life Technologies) and doxycycline (2 μg/ml: Sigma Aldrich). Medium was changed every other day. Differentiating iPSC-iNs were purified by gating on eGFP+/PSA-NCAM+ neurons at 3, 7 and 14 days after conversion. 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). ENA first public 2018-01-01 ENA last update 2017-08-25 External Id SAMEA104227990 INSDC center alias Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC center name Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC first public 2018-01-01T17:01:42Z INSDC last update 2017-08-25T17:29:35Z INSDC status public Submitter Id E-MTAB-6018:Clue_iPSC_iN3d.fastq.gz broker name ArrayExpress cell type neuron common name human disease normal genotype pLV-X-Ngn2-2A-eGFP (N2AG) individual Clue organism part skin phenotype Ngn2+/PSA-NCAM+ progenitor cell type induced pluripotent stem cell sample name E-MTAB-6018:Clue_iPSC_iN3d.fastq.gz time 3 ERS1887011 SAMEA104227993 9606 Homo sapiens Protocols: 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). ENA first public 2018-01-01 ENA last update 2017-08-25 External Id SAMEA104227993 INSDC center alias Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC center name Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC first public 2018-01-01T17:01:42Z INSDC last update 2017-08-25T17:29:35Z INSDC status public Submitter Id E-MTAB-6018:Cove_D2.fastq.gz broker name ArrayExpress cell type neuron common name human disease normal genotype pRV-CAG:LckN-eGFP individual Cove organism part skin phenotype Ngn2+/PSA-NCAM+ progenitor cell type neuronal stem cell sample name E-MTAB-6018:Cove_D2.fastq.gz time 2 ERS1887013 SAMEA104227995 9606 Homo sapiens Protocols: 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). ENA first public 2018-01-01 ENA last update 2017-08-25 External Id SAMEA104227995 INSDC center alias Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC center name Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC first public 2018-01-01T17:01:42Z INSDC last update 2017-08-25T17:29:35Z INSDC status public Submitter Id E-MTAB-6018:Cove_D7.fastq.gz broker name ArrayExpress cell type neuron common name human disease normal genotype pRV-CAG:LckN-eGFP individual Cove organism part skin phenotype Ngn2+/PSA-NCAM+ progenitor cell type neuronal stem cell sample name E-MTAB-6018:Cove_D7.fastq.gz time 7 ERS1887014 SAMEA104227996 9606 Homo sapiens Protocols: 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. Induced iPSC neurons (iPSC-iNs): The cell surface markers SSEA4+ and TRA1-81+ were used for FACS-based purification of iPSCs. For iPSC-iNs, the human cDNA sequence for Ngn2 and the sequence for eGFP were linked by a 2A sequence and cloned into the tet-inducible pLV-XTP backbone (Clonetech). IPSCs were transduced with lentiviral particles for pLV-X-UbC:TetOn (UtO) and pLV-X-Ngn2:2A:eGFP (N2AG) and were selected in the presence of puromycin (0.5 μg/ml, Sigma Aldrich) and G418 (100 μg/ml, Life technologies). For iPSC-iN conversion, iPSCs were transferred into PluriPro monolayer conditions (Cell Guidance Systems) and, 24 hrs after plating, were induced with doxycycline (2 μg/ml: Sigma Aldrich) for two days. For further maturation, iNs were switched to Neuron Maturation Media (NMM) based on DMEM:F12/Neurobasal (both Gibco) (1:1) as used for directed differentiation. NMM contains the following supplements: N2 supplement (1x, Gibco), B27 supplement (1x, Gibco), Laminin (1 μg/ml, Life Technologies) and doxycycline (2 μg/ml: Sigma Aldrich). Medium was changed every other day. Differentiating iPSC-iNs were purified by gating on eGFP+/PSA-NCAM+ neurons at 3, 7 and 14 days after conversion. 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). ENA first public 2018-01-01 ENA last update 2017-08-25 External Id SAMEA104227996 INSDC center alias Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC center name Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC first public 2018-01-01T17:01:42Z INSDC last update 2017-08-25T17:29:35Z INSDC status public Submitter Id E-MTAB-6018:Cove_iPSC_iN14d.fastq.gz broker name ArrayExpress cell type neuron common name human disease normal genotype pLV-X-Ngn2-2A-eGFP (N2AG) individual Cove organism part skin phenotype Ngn2+/PSA-NCAM+ progenitor cell type induced pluripotent stem cell sample name E-MTAB-6018:Cove_iPSC_iN14d.fastq.gz time 14 ERS1887015 SAMEA104227997 9606 Homo sapiens Protocols: 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. Induced iPSC neurons (iPSC-iNs): The cell surface markers SSEA4+ and TRA1-81+ were used for FACS-based purification of iPSCs. For iPSC-iNs, the human cDNA sequence for Ngn2 and the sequence for eGFP were linked by a 2A sequence and cloned into the tet-inducible pLV-XTP backbone (Clonetech). IPSCs were transduced with lentiviral particles for pLV-X-UbC:TetOn (UtO) and pLV-X-Ngn2:2A:eGFP (N2AG) and were selected in the presence of puromycin (0.5 μg/ml, Sigma Aldrich) and G418 (100 μg/ml, Life technologies). For iPSC-iN conversion, iPSCs were transferred into PluriPro monolayer conditions (Cell Guidance Systems) and, 24 hrs after plating, were induced with doxycycline (2 μg/ml: Sigma Aldrich) for two days. For further maturation, iNs were switched to Neuron Maturation Media (NMM) based on DMEM:F12/Neurobasal (both Gibco) (1:1) as used for directed differentiation. NMM contains the following supplements: N2 supplement (1x, Gibco), B27 supplement (1x, Gibco), Laminin (1 μg/ml, Life Technologies) and doxycycline (2 μg/ml: Sigma Aldrich). Medium was changed every other day. Differentiating iPSC-iNs were purified by gating on eGFP+/PSA-NCAM+ neurons at 3, 7 and 14 days after conversion. 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). ENA first public 2018-01-01 ENA last update 2017-08-25 External Id SAMEA104227997 INSDC center alias Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC center name Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC first public 2018-01-01T17:01:42Z INSDC last update 2017-08-25T17:29:35Z INSDC status public Submitter Id E-MTAB-6018:Cove_iPSC_iN3d.fastq.gz broker name ArrayExpress cell type neuron common name human disease normal genotype pLV-X-Ngn2-2A-eGFP (N2AG) individual Cove organism part skin phenotype Ngn2+/PSA-NCAM+ progenitor cell type induced pluripotent stem cell sample name E-MTAB-6018:Cove_iPSC_iN3d.fastq.gz time 3 ERS1887016 SAMEA104227998 9606 Homo sapiens Protocols: 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. Induced iPSC neurons (iPSC-iNs): The cell surface markers SSEA4+ and TRA1-81+ were used for FACS-based purification of iPSCs. For iPSC-iNs, the human cDNA sequence for Ngn2 and the sequence for eGFP were linked by a 2A sequence and cloned into the tet-inducible pLV-XTP backbone (Clonetech). IPSCs were transduced with lentiviral particles for pLV-X-UbC:TetOn (UtO) and pLV-X-Ngn2:2A:eGFP (N2AG) and were selected in the presence of puromycin (0.5 μg/ml, Sigma Aldrich) and G418 (100 μg/ml, Life technologies). For iPSC-iN conversion, iPSCs were transferred into PluriPro monolayer conditions (Cell Guidance Systems) and, 24 hrs after plating, were induced with doxycycline (2 μg/ml: Sigma Aldrich) for two days. For further maturation, iNs were switched to Neuron Maturation Media (NMM) based on DMEM:F12/Neurobasal (both Gibco) (1:1) as used for directed differentiation. NMM contains the following supplements: N2 supplement (1x, Gibco), B27 supplement (1x, Gibco), Laminin (1 μg/ml, Life Technologies) and doxycycline (2 μg/ml: Sigma Aldrich). Medium was changed every other day. Differentiating iPSC-iNs were purified by gating on eGFP+/PSA-NCAM+ neurons at 3, 7 and 14 days after conversion. 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). ENA first public 2018-01-01 ENA last update 2017-08-25 External Id SAMEA104227998 INSDC center alias Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC center name Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC first public 2018-01-01T17:01:42Z INSDC last update 2017-08-25T17:29:35Z INSDC status public Submitter Id E-MTAB-6018:Cove_iPSC_iN7d.fastq.gz broker name ArrayExpress cell type neuron common name human disease normal genotype pLV-X-Ngn2-2A-eGFP (N2AG) individual Cove organism part skin phenotype Ngn2+/PSA-NCAM+ progenitor cell type induced pluripotent stem cell sample name E-MTAB-6018:Cove_iPSC_iN7d.fastq.gz time 7 ERS1887018 SAMEA104228000 9606 Homo sapiens Protocols: 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. Induced iPSC neurons (iPSC-iNs): The cell surface markers SSEA4+ and TRA1-81+ were used for FACS-based purification of iPSCs. For iPSC-iNs, the human cDNA sequence for Ngn2 and the sequence for eGFP were linked by a 2A sequence and cloned into the tet-inducible pLV-XTP backbone (Clonetech). IPSCs were transduced with lentiviral particles for pLV-X-UbC:TetOn (UtO) and pLV-X-Ngn2:2A:eGFP (N2AG) and were selected in the presence of puromycin (0.5 μg/ml, Sigma Aldrich) and G418 (100 μg/ml, Life technologies). For iPSC-iN conversion, iPSCs were transferred into PluriPro monolayer conditions (Cell Guidance Systems) and, 24 hrs after plating, were induced with doxycycline (2 μg/ml: Sigma Aldrich) for two days. For further maturation, iNs were switched to Neuron Maturation Media (NMM) based on DMEM:F12/Neurobasal (both Gibco) (1:1) as used for directed differentiation. NMM contains the following supplements: N2 supplement (1x, Gibco), B27 supplement (1x, Gibco), Laminin (1 μg/ml, Life Technologies) and doxycycline (2 μg/ml: Sigma Aldrich). Medium was changed every other day. Differentiating iPSC-iNs were purified by gating on eGFP+/PSA-NCAM+ neurons at 3, 7 and 14 days after conversion. 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). ENA first public 2018-01-01 ENA last update 2017-08-25 External Id SAMEA104228000 INSDC center alias Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC center name Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC first public 2018-01-01T17:01:42Z INSDC last update 2017-08-25T17:29:35Z INSDC status public Submitter Id E-MTAB-6018:Acai_iPSC.fastq.gz broker name ArrayExpress cell type induced pluripotent stem cell common name human disease autism spectrum disorder genotype pLV-X-Ngn2-2A-eGFP (N2AG) individual Acai organism part skin phenotype SSEA-4+/TRA1-81+ progenitor cell type fibroblast of dermis sample name E-MTAB-6018:Acai_iPSC.fastq.gz ERS1887020 SAMEA104228002 9606 Homo sapiens Protocols: 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. Induced iPSC neurons (iPSC-iNs): The cell surface markers SSEA4+ and TRA1-81+ were used for FACS-based purification of iPSCs. For iPSC-iNs, the human cDNA sequence for Ngn2 and the sequence for eGFP were linked by a 2A sequence and cloned into the tet-inducible pLV-XTP backbone (Clonetech). IPSCs were transduced with lentiviral particles for pLV-X-UbC:TetOn (UtO) and pLV-X-Ngn2:2A:eGFP (N2AG) and were selected in the presence of puromycin (0.5 μg/ml, Sigma Aldrich) and G418 (100 μg/ml, Life technologies). For iPSC-iN conversion, iPSCs were transferred into PluriPro monolayer conditions (Cell Guidance Systems) and, 24 hrs after plating, were induced with doxycycline (2 μg/ml: Sigma Aldrich) for two days. For further maturation, iNs were switched to Neuron Maturation Media (NMM) based on DMEM:F12/Neurobasal (both Gibco) (1:1) as used for directed differentiation. NMM contains the following supplements: N2 supplement (1x, Gibco), B27 supplement (1x, Gibco), Laminin (1 μg/ml, Life Technologies) and doxycycline (2 μg/ml: Sigma Aldrich). Medium was changed every other day. Differentiating iPSC-iNs were purified by gating on eGFP+/PSA-NCAM+ neurons at 3, 7 and 14 days after conversion. 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). ENA first public 2018-01-01 ENA last update 2017-08-25 External Id SAMEA104228002 INSDC center alias Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC center name Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC first public 2018-01-01T17:01:42Z INSDC last update 2017-08-25T17:29:35Z INSDC status public Submitter Id E-MTAB-6018:Agua_iPSC.fastq.gz broker name ArrayExpress cell type induced pluripotent stem cell common name human disease autism spectrum disorder genotype pLV-X-Ngn2-2A-eGFP (N2AG) individual Agua organism part skin phenotype SSEA-4+/TRA1-81+ progenitor cell type fibroblast of dermis sample name E-MTAB-6018:Agua_iPSC.fastq.gz ERS1887022 SAMEA104228004 9606 Homo sapiens Protocols: 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. Induced iPSC neurons (iPSC-iNs): The cell surface markers SSEA4+ and TRA1-81+ were used for FACS-based purification of iPSCs. For iPSC-iNs, the human cDNA sequence for Ngn2 and the sequence for eGFP were linked by a 2A sequence and cloned into the tet-inducible pLV-XTP backbone (Clonetech). IPSCs were transduced with lentiviral particles for pLV-X-UbC:TetOn (UtO) and pLV-X-Ngn2:2A:eGFP (N2AG) and were selected in the presence of puromycin (0.5 μg/ml, Sigma Aldrich) and G418 (100 μg/ml, Life technologies). For iPSC-iN conversion, iPSCs were transferred into PluriPro monolayer conditions (Cell Guidance Systems) and, 24 hrs after plating, were induced with doxycycline (2 μg/ml: Sigma Aldrich) for two days. For further maturation, iNs were switched to Neuron Maturation Media (NMM) based on DMEM:F12/Neurobasal (both Gibco) (1:1) as used for directed differentiation. NMM contains the following supplements: N2 supplement (1x, Gibco), B27 supplement (1x, Gibco), Laminin (1 μg/ml, Life Technologies) and doxycycline (2 μg/ml: Sigma Aldrich). Medium was changed every other day. Differentiating iPSC-iNs were purified by gating on eGFP+/PSA-NCAM+ neurons at 3, 7 and 14 days after conversion. 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). ENA first public 2018-01-01 ENA last update 2017-08-25 External Id SAMEA104228004 INSDC center alias Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC center name Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC first public 2018-01-01T17:01:42Z INSDC last update 2017-08-25T17:29:35Z INSDC status public Submitter Id E-MTAB-6018:Apex_iPSC.fastq.gz broker name ArrayExpress cell type induced pluripotent stem cell common name human disease autism spectrum disorder genotype pLV-X-Ngn2-2A-eGFP (N2AG) individual Apex organism part skin phenotype SSEA-4+/TRA1-81+ progenitor cell type fibroblast of dermis sample name E-MTAB-6018:Apex_iPSC.fastq.gz ERS1887023 SAMEA104228005 9606 Homo sapiens Protocols: 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. Induced iPSC neurons (iPSC-iNs): The cell surface markers SSEA4+ and TRA1-81+ were used for FACS-based purification of iPSCs. For iPSC-iNs, the human cDNA sequence for Ngn2 and the sequence for eGFP were linked by a 2A sequence and cloned into the tet-inducible pLV-XTP backbone (Clonetech). IPSCs were transduced with lentiviral particles for pLV-X-UbC:TetOn (UtO) and pLV-X-Ngn2:2A:eGFP (N2AG) and were selected in the presence of puromycin (0.5 μg/ml, Sigma Aldrich) and G418 (100 μg/ml, Life technologies). For iPSC-iN conversion, iPSCs were transferred into PluriPro monolayer conditions (Cell Guidance Systems) and, 24 hrs after plating, were induced with doxycycline (2 μg/ml: Sigma Aldrich) for two days. For further maturation, iNs were switched to Neuron Maturation Media (NMM) based on DMEM:F12/Neurobasal (both Gibco) (1:1) as used for directed differentiation. NMM contains the following supplements: N2 supplement (1x, Gibco), B27 supplement (1x, Gibco), Laminin (1 μg/ml, Life Technologies) and doxycycline (2 μg/ml: Sigma Aldrich). Medium was changed every other day. Differentiating iPSC-iNs were purified by gating on eGFP+/PSA-NCAM+ neurons at 3, 7 and 14 days after conversion. 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). ENA first public 2018-01-01 ENA last update 2017-08-25 External Id SAMEA104228005 INSDC center alias Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC center name Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC first public 2018-01-01T17:01:42Z INSDC last update 2017-08-25T17:29:35Z INSDC status public Submitter Id E-MTAB-6018:Arch_iPSC.fastq.gz broker name ArrayExpress cell type induced pluripotent stem cell common name human disease autism spectrum disorder genotype pLV-X-Ngn2-2A-eGFP (N2AG) individual Arch organism part skin phenotype SSEA-4+/TRA1-81+ progenitor cell type fibroblast of dermis sample name E-MTAB-6018:Arch_iPSC.fastq.gz ERS1887024 SAMEA104228006 9606 Homo sapiens Protocols: 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. Induced iPSC neurons (iPSC-iNs): The cell surface markers SSEA4+ and TRA1-81+ were used for FACS-based purification of iPSCs. For iPSC-iNs, the human cDNA sequence for Ngn2 and the sequence for eGFP were linked by a 2A sequence and cloned into the tet-inducible pLV-XTP backbone (Clonetech). IPSCs were transduced with lentiviral particles for pLV-X-UbC:TetOn (UtO) and pLV-X-Ngn2:2A:eGFP (N2AG) and were selected in the presence of puromycin (0.5 μg/ml, Sigma Aldrich) and G418 (100 μg/ml, Life technologies). For iPSC-iN conversion, iPSCs were transferred into PluriPro monolayer conditions (Cell Guidance Systems) and, 24 hrs after plating, were induced with doxycycline (2 μg/ml: Sigma Aldrich) for two days. For further maturation, iNs were switched to Neuron Maturation Media (NMM) based on DMEM:F12/Neurobasal (both Gibco) (1:1) as used for directed differentiation. NMM contains the following supplements: N2 supplement (1x, Gibco), B27 supplement (1x, Gibco), Laminin (1 μg/ml, Life Technologies) and doxycycline (2 μg/ml: Sigma Aldrich). Medium was changed every other day. Differentiating iPSC-iNs were purified by gating on eGFP+/PSA-NCAM+ neurons at 3, 7 and 14 days after conversion. 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). ENA first public 2018-01-01 ENA last update 2017-08-25 External Id SAMEA104228006 INSDC center alias Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC center name Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC first public 2018-01-01T17:01:42Z INSDC last update 2017-08-25T17:29:35Z INSDC status public Submitter Id E-MTAB-6018:Avid_iPSC.fastq.gz broker name ArrayExpress cell type induced pluripotent stem cell common name human disease autism spectrum disorder genotype pLV-X-Ngn2-2A-eGFP (N2AG) individual Avid organism part skin phenotype SSEA-4+/TRA1-81+ progenitor cell type fibroblast of dermis sample name E-MTAB-6018:Avid_iPSC.fastq.gz ERS1887025 SAMEA104228007 9606 Homo sapiens Protocols: 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. Induced iPSC neurons (iPSC-iNs): The cell surface markers SSEA4+ and TRA1-81+ were used for FACS-based purification of iPSCs. For iPSC-iNs, the human cDNA sequence for Ngn2 and the sequence for eGFP were linked by a 2A sequence and cloned into the tet-inducible pLV-XTP backbone (Clonetech). IPSCs were transduced with lentiviral particles for pLV-X-UbC:TetOn (UtO) and pLV-X-Ngn2:2A:eGFP (N2AG) and were selected in the presence of puromycin (0.5 μg/ml, Sigma Aldrich) and G418 (100 μg/ml, Life technologies). For iPSC-iN conversion, iPSCs were transferred into PluriPro monolayer conditions (Cell Guidance Systems) and, 24 hrs after plating, were induced with doxycycline (2 μg/ml: Sigma Aldrich) for two days. For further maturation, iNs were switched to Neuron Maturation Media (NMM) based on DMEM:F12/Neurobasal (both Gibco) (1:1) as used for directed differentiation. NMM contains the following supplements: N2 supplement (1x, Gibco), B27 supplement (1x, Gibco), Laminin (1 μg/ml, Life Technologies) and doxycycline (2 μg/ml: Sigma Aldrich). Medium was changed every other day. Differentiating iPSC-iNs were purified by gating on eGFP+/PSA-NCAM+ neurons at 3, 7 and 14 days after conversion. 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). ENA first public 2018-01-01 ENA last update 2017-08-25 External Id SAMEA104228007 INSDC center alias Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC center name Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC first public 2018-01-01T17:01:42Z INSDC last update 2017-08-25T17:29:35Z INSDC status public Submitter Id E-MTAB-6018:Cent_iPSC.fastq.gz broker name ArrayExpress cell type induced pluripotent stem cell common name human disease normal genotype pLV-X-Ngn2-2A-eGFP (N2AG) individual Cent organism part skin phenotype SSEA-4+/TRA1-81+ progenitor cell type fibroblast of dermis sample name E-MTAB-6018:Cent_iPSC.fastq.gz ERS1887026 SAMEA104228008 9606 Homo sapiens Protocols: 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. Induced iPSC neurons (iPSC-iNs): The cell surface markers SSEA4+ and TRA1-81+ were used for FACS-based purification of iPSCs. For iPSC-iNs, the human cDNA sequence for Ngn2 and the sequence for eGFP were linked by a 2A sequence and cloned into the tet-inducible pLV-XTP backbone (Clonetech). IPSCs were transduced with lentiviral particles for pLV-X-UbC:TetOn (UtO) and pLV-X-Ngn2:2A:eGFP (N2AG) and were selected in the presence of puromycin (0.5 μg/ml, Sigma Aldrich) and G418 (100 μg/ml, Life technologies). For iPSC-iN conversion, iPSCs were transferred into PluriPro monolayer conditions (Cell Guidance Systems) and, 24 hrs after plating, were induced with doxycycline (2 μg/ml: Sigma Aldrich) for two days. For further maturation, iNs were switched to Neuron Maturation Media (NMM) based on DMEM:F12/Neurobasal (both Gibco) (1:1) as used for directed differentiation. NMM contains the following supplements: N2 supplement (1x, Gibco), B27 supplement (1x, Gibco), Laminin (1 μg/ml, Life Technologies) and doxycycline (2 μg/ml: Sigma Aldrich). Medium was changed every other day. Differentiating iPSC-iNs were purified by gating on eGFP+/PSA-NCAM+ neurons at 3, 7 and 14 days after conversion. 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). ENA first public 2018-01-01 ENA last update 2017-08-25 External Id SAMEA104228008 INSDC center alias Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC center name Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC first public 2018-01-01T17:01:42Z INSDC last update 2017-08-25T17:29:35Z INSDC status public Submitter Id E-MTAB-6018:Chap_iPSC.fastq.gz broker name ArrayExpress cell type induced pluripotent stem cell common name human disease normal genotype pLV-X-Ngn2-2A-eGFP (N2AG) individual Chap organism part skin phenotype SSEA-4+/TRA1-81+ progenitor cell type fibroblast of dermis sample name E-MTAB-6018:Chap_iPSC.fastq.gz ERS1887027 SAMEA104228009 9606 Homo sapiens Protocols: 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. Induced iPSC neurons (iPSC-iNs): The cell surface markers SSEA4+ and TRA1-81+ were used for FACS-based purification of iPSCs. For iPSC-iNs, the human cDNA sequence for Ngn2 and the sequence for eGFP were linked by a 2A sequence and cloned into the tet-inducible pLV-XTP backbone (Clonetech). IPSCs were transduced with lentiviral particles for pLV-X-UbC:TetOn (UtO) and pLV-X-Ngn2:2A:eGFP (N2AG) and were selected in the presence of puromycin (0.5 μg/ml, Sigma Aldrich) and G418 (100 μg/ml, Life technologies). For iPSC-iN conversion, iPSCs were transferred into PluriPro monolayer conditions (Cell Guidance Systems) and, 24 hrs after plating, were induced with doxycycline (2 μg/ml: Sigma Aldrich) for two days. For further maturation, iNs were switched to Neuron Maturation Media (NMM) based on DMEM:F12/Neurobasal (both Gibco) (1:1) as used for directed differentiation. NMM contains the following supplements: N2 supplement (1x, Gibco), B27 supplement (1x, Gibco), Laminin (1 μg/ml, Life Technologies) and doxycycline (2 μg/ml: Sigma Aldrich). Medium was changed every other day. Differentiating iPSC-iNs were purified by gating on eGFP+/PSA-NCAM+ neurons at 3, 7 and 14 days after conversion. 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). ENA first public 2018-01-01 ENA last update 2017-08-25 External Id SAMEA104228009 INSDC center alias Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC center name Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC first public 2018-01-01T17:01:42Z INSDC last update 2017-08-25T17:29:35Z INSDC status public Submitter Id E-MTAB-6018:Clay_iPSC.fastq.gz broker name ArrayExpress cell type induced pluripotent stem cell common name human disease normal genotype pLV-X-Ngn2-2A-eGFP (N2AG) individual Clay organism part skin phenotype SSEA-4+/TRA1-81+ progenitor cell type fibroblast of dermis sample name E-MTAB-6018:Clay_iPSC.fastq.gz ERS1887028 SAMEA104228010 9606 Homo sapiens Protocols: 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. Induced iPSC neurons (iPSC-iNs): The cell surface markers SSEA4+ and TRA1-81+ were used for FACS-based purification of iPSCs. For iPSC-iNs, the human cDNA sequence for Ngn2 and the sequence for eGFP were linked by a 2A sequence and cloned into the tet-inducible pLV-XTP backbone (Clonetech). IPSCs were transduced with lentiviral particles for pLV-X-UbC:TetOn (UtO) and pLV-X-Ngn2:2A:eGFP (N2AG) and were selected in the presence of puromycin (0.5 μg/ml, Sigma Aldrich) and G418 (100 μg/ml, Life technologies). For iPSC-iN conversion, iPSCs were transferred into PluriPro monolayer conditions (Cell Guidance Systems) and, 24 hrs after plating, were induced with doxycycline (2 μg/ml: Sigma Aldrich) for two days. For further maturation, iNs were switched to Neuron Maturation Media (NMM) based on DMEM:F12/Neurobasal (both Gibco) (1:1) as used for directed differentiation. NMM contains the following supplements: N2 supplement (1x, Gibco), B27 supplement (1x, Gibco), Laminin (1 μg/ml, Life Technologies) and doxycycline (2 μg/ml: Sigma Aldrich). Medium was changed every other day. Differentiating iPSC-iNs were purified by gating on eGFP+/PSA-NCAM+ neurons at 3, 7 and 14 days after conversion. 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). ENA first public 2018-01-01 ENA last update 2017-08-25 External Id SAMEA104228010 INSDC center alias Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC center name Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC first public 2018-01-01T17:01:42Z INSDC last update 2017-08-25T17:29:35Z INSDC status public Submitter Id E-MTAB-6018:Clue_iPSC.fastq.gz broker name ArrayExpress cell type induced pluripotent stem cell common name human disease normal genotype pLV-X-Ngn2-2A-eGFP (N2AG) individual Clue organism part skin phenotype SSEA-4+/TRA1-81+ progenitor cell type fibroblast of dermis sample name E-MTAB-6018:Clue_iPSC.fastq.gz ERS1887029 SAMEA104228011 9606 Homo sapiens Protocols: 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. Induced iPSC neurons (iPSC-iNs): The cell surface markers SSEA4+ and TRA1-81+ were used for FACS-based purification of iPSCs. For iPSC-iNs, the human cDNA sequence for Ngn2 and the sequence for eGFP were linked by a 2A sequence and cloned into the tet-inducible pLV-XTP backbone (Clonetech). IPSCs were transduced with lentiviral particles for pLV-X-UbC:TetOn (UtO) and pLV-X-Ngn2:2A:eGFP (N2AG) and were selected in the presence of puromycin (0.5 μg/ml, Sigma Aldrich) and G418 (100 μg/ml, Life technologies). For iPSC-iN conversion, iPSCs were transferred into PluriPro monolayer conditions (Cell Guidance Systems) and, 24 hrs after plating, were induced with doxycycline (2 μg/ml: Sigma Aldrich) for two days. For further maturation, iNs were switched to Neuron Maturation Media (NMM) based on DMEM:F12/Neurobasal (both Gibco) (1:1) as used for directed differentiation. NMM contains the following supplements: N2 supplement (1x, Gibco), B27 supplement (1x, Gibco), Laminin (1 μg/ml, Life Technologies) and doxycycline (2 μg/ml: Sigma Aldrich). Medium was changed every other day. Differentiating iPSC-iNs were purified by gating on eGFP+/PSA-NCAM+ neurons at 3, 7 and 14 days after conversion. 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). ENA first public 2018-01-01 ENA last update 2017-08-25 External Id SAMEA104228011 INSDC center alias Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC center name Laboratory of Genetics - Gage The Salk Institute for Biological Studies INSDC first public 2018-01-01T17:01:42Z INSDC last update 2017-08-25T17:29:36Z INSDC status public Submitter Id E-MTAB-6018:Cove_iPSC.fastq.gz broker name ArrayExpress cell type induced pluripotent stem cell common name human disease normal genotype pLV-X-Ngn2-2A-eGFP (N2AG) individual Cove organism part skin phenotype SSEA-4+/TRA1-81+ progenitor cell type fibroblast of dermis sample name E-MTAB-6018:Cove_iPSC.fastq.gz