RNA-seq of AD patient-derived induced neurons treated with shikonin
RNA-seq of AD patient-derived induced neurons treated with shikonin
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).