RNA-seq of subcellular neuronal compartments
RNA-seq of subcellular neuronal compartments
hiPSCs with NGN2, ISL1, and LHX3 cassettes were differentiated into induced motor neurons (iMNs) using established protocols (Fernandopulle et al., 2018, doi: 10.1002/cpcb.51). Cells were grown on filter membrane inserts (pore size 1.0 μm PET membrane for 6-well plates Millicell PLRP06H48, Millipore) to isolate axonal and somatodendritic compartments. Isolation was performed on day 21 of the differentiation protocol. RNA was extracted from using TRIfast (PeqLab 30-2010). For a detailed description of harvesting material from filter membranes see our prior work (Ludwik et al., 2019, doi: 10.1016/j.ymeth.2019.02.002).  100 ng of total RNA obtained from either total cells or separated compartments was used for library preparation with the Truseq stranded total library prep kit (Illumina 20020596) according to the manufacturer's recommendation.