DR3
Homo sapiens
Protocols: RNA was isolated from 12 NPC lines (8 control and 4 case lines). All iPSCs were derived from human donor skin fibroblasts using integration-free episomal methods. Plasmids incorporating Oct4/shP53, SOX2/Klf4 and L-Myc/Lin28 (pCXLE-hSK and pCXLE-hUL, Addgene plasmids 27077, 27078, 27080) were electroporated into dermal fibroblasts using an Amaxa Nucleofection Kit (Lonza). Colonies were selected between day 21-25 and individual colonies of cells were expanded into single wells coated with Geltrex (Life Technologies) in Essential 8 media (Life Technologies) at 37C and 5% CO2. Prior to neuralisation iPSC were transitioned to reduced-growth factor Matrigel (BD Biosciences) coated plastic dishes. Anterior NPCs were then derived from parental pluripotent stem cell lines. iPS cells were neurally converted in suspension in CDM as described in Bilican et al. \Physiological Normoxia and Absence of EGF Is Required for the Long-Term Propagation of Anterior Neural Precursors from Human Pluripotent Cells\, PLoS One (2014), 9(1): e85932. RNA was extracted from cell pellets using the RNeasy Mini kit (QIAGEN). Genomic DNA was removed with the DNA-Free kit (Ambion). QC analysis using an Agilent 2100 bioanalyzer demonstrated that all samples had RNA integrity number (RIN) values between 7.7-10. Libraries were prepared with the TruSeq Stranded Total RNA with Ribo-Zero Gold kit.