E-MTAB-6018:Acai_iPSC_iN3d.fastq.gz_s
Illumina HiSeq 2500 sequencing; Time line RNAseq of iPSC-derived neurons from ASD and control subjects.
Time line RNAseq of iPSC-derived neurons from ASD and control subjects.
Acai_iPSC_iN3d.fastq.gz_s
We first used fluorescence-activated cell sorting (FACS) to purify a homogeneous population of NSCs (D0) based on the expression of the cell-surface markers CD184+/CD271-/CD44-/CD24-/CD15+. To trace ASD and control neurons over time, we performed a series of retroviral lineage-tracing experiments to trace the progenies of dividing NSCs using a retroviral vector expressing a membrane-tagged enhanced green fluorescent protein (eGFP) (CAG::LckN-eGFP). As differentiating neurons express PSA-NCAM on the cell surface, we established a FACS-based protocol for purification of defined subpopulations of retrovirally labeled eGFP+/PSA-NCAM+ double-positive neurons after 2, 4, 7 and 14 (D2, 4, 7 and 14) days of differentiation. IPSCs were sorted based on the expression of SSEA-4 and TRA1-81 and maturing iPSC-iNs were collected at the indicated days after induction by sorting for eGFP (indicative for the Ngn2 transgene)- and PSA-NCAM-positive cells. 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).
Laboratory of Genetics - Gage The Salk Institute for Biological Studies
E-MTAB-6018:Acai_iPSC_iN3d.fastq.gz_s
Experimental Factor: disease
autism spectrum disorder
Experimental Factor: progenitor cell type
induced pluripotent stem cell
Experimental Factor: cell type
neuron
Experimental Factor: time
3