Total RNA was extracted from differentiated primary myoblasts using the RNeasy mini kit (QIAGEN). To prepare the cDNA library, 10 ng of total RNA was diluted with 9 µL of RNase free water, then mixed with VN primer (Oxford NANOPORE Technologies, UK), 1 µL of 10 mM dNTPs (New England Biolabs Inc. Ipswich, Massachusetts, USA), and incubated at 65˚C for 5 min as the RNA solution. Separately, 4 µL of 5x RT Buffer (Thermo Fisher Scientific), 1 µL of RNaseOUT (Thermo Fisher Scientific), 1 µL of Nuclease-free water and 2 µL of Strand-Switching Primer (Oxford NANOPORE Technologies) was mixed as the strand-switching buffer. The two solutions were mixed at 42˚C for 2 min; then, 1 µL of Maxima H Minus Reverse Transcriptase (Thermo Fisher Scientific) was added, incubated at 42˚C for 90 min, 85˚C for 5 min, and stored at 4˚C until use as the cDNA library.
GEO Accession
GSM4585063