Following 3 weeks of iN conversion, iNs were detached using TrypLE and stained for PSA-NCAM (1:100) for 45 min at 4°C in sorting buffer (250 mM myo-inositol and 5 μg/ml polyvinyl alcohol, PVA, in PBS) containing 1% KOSR. Cells were washed and stained with Alexa-747 anti mouse IgM for 30 min at 4°C, resuspended in sorting buffer containing EDTA and DNase, and filtered using a 40 μm cell strainer. The Alexa647-positive population was sorted directly into Trizol-LS, and RNA was isolated according to the manufacturer’s instructions and digested with TURBO DNase (Life Technologies). RNA integrity (RIN) numbers were assessed using the Agilent TapeStation before library preparation.
