title: AD7 S
organism: Homo sapiens
description: Protocols: For RNA-Seq, total bulk RNA was extracted from flow cytometry-isolated iNs following 24 days of conversion. iNs were sorted based on the expression of the PSA-NCAM surface markers using PSA-NCAM-PE (Miltenyi). Sorting was performed at 4 °C directly into ice-cold Trizol LS reagent (ThermoFisher). Human fibroblasts were obtained from the UCSD Shiley-Marcos Alzheimer's disease research center (ADRC) and provided written informed consent. All procedures were approved by local human subjects committees. Fibroblasts were cultured in DMEM containing 15 % fetal bovine serum and 0.1 % non-essential amino acids at 37°C with 5 % CO2. They were transduced with a lentivirus pLVXUbC-rtTA-Ngn2:2A:Ascl1 (Addgene: #127289) and selected with puromycin (1 µg/ml) as previously described (Mertens et al., 2021). To initiate conversion, puromycin-selected fibroblasts were pooled 3:1 and, after 24 hours, medium was changed to neural conversion medium for three weeks. Neural conversion medium is based on DMEM:F12 and Neurobasal (1:1), supplemented with N2 supplement (1x, ThermoFisher), B27 supplement (1x, ThermoFisher), doxycycline (2 µg/ml, Sigma Aldrich), Laminin (1 µg/ml, Sigma Aldrich), dibutyryl-cyclic-AMP (100 µg/ml, Santa Cruz), human recombinant noggin (150 ng/ml, R&D), LDN-193189 (0.5 µM, Sanova Pharma), A83-1 (0.5 µM, Santa Cruz), CHIR99021 (3 µM, LC Laboratories), forskoling (5 µM, LC Laboratories) and SB-431542 (10 µM, MedChem). After two weeks of conversion, cells were treated for 10 days with shikonin (10 µM, ChemCruz) before FACS sorting and harvest of RNA in Trizol. RNA was extracted according to the manufacturer's protocol, followed by TURBO DNase digestion (ThermoFisher). RNA integrity was assessed using the Bioanalyzer High Sensitivity RNA Analysis Kit (Agilent). A cDNA library was generated using the TruSeq Stranded mRNA Sample Preparation Kit (Illumina).
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2022-07-05
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2022-07-05
attributes.tag: External Id
attributes.value: SAMEA110175488
attributes.tag: INSDC center alias
attributes.value: University of Innsbruck, Austria Salk Institute
attributes.tag: INSDC center name
attributes.value: University of Innsbruck, Austria Salk Institute
attributes.tag: INSDC first public
attributes.value: 2022-07-05T12:03:59Z
attributes.tag: INSDC last update
attributes.value: 2022-07-05T12:03:59Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-11855:AD7 S
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell_type
attributes.value: neuron
attributes.tag: common name
attributes.value: human
attributes.tag: developmental stage
attributes.value: adult
attributes.tag: disease
attributes.value: Alzheimer disease
attributes.tag: genotype
attributes.value: somatic genotype
attributes.tag: individual
attributes.value: AD7
attributes.tag: isolate
attributes.value: not applicable
attributes.tag: organism part
attributes.value: skin of body
attributes.tag: progenitor cell type
attributes.value: fibroblast
attributes.tag: sample name
attributes.value: E-MTAB-11855:AD7 S