RNA-seq of subcellular neuronal compartments of ALS model
RNA-seq of subcellular neuronal compartments of ALS model
Pateint-derived iPSC with FUS(R244RR) and isogenic line 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. Whole iMNs and subcellular compartments were collected 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 mRNA library prep kit (Illumina 20020595) according to the manufacturer's recommendation.