Total RNA from ESCs, EBs, 293T cells, MBs, and MTs was isolated with RNeasy (QIAGEN) and reverse transcribed using SuperScript III and random hexamers (Life Technologies) to synthesize the first strand. Second strand was synthesized with deoxyuridine triphosphate to generate strand asymmetry using DNA polymerase I [M0209L, New England Biolabs (NEB)] and the Escherichia coli Ligase (L6090L, Enzymatics). RNA-seq libraries were constructed using the protocol described in (43).

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