Truseq stranded total rna with ribo zero gold protocol

Total RNA from myotube cultures was isolated with Trizol Reagent using a kit (Life Technologies; Cat #15596-018,) and extracted using a miRNAeasy kit (Qiagen; Cat #217004).
Total RNA concentration was quantified using a NanoDrop ND-1000 Spectrophotometer (Thermo Fisher Scientific, Waltham, MA). Aliquots of RNA (1 µg) were processed using the Illumina TruSeq Stranded Total RNA with Ribo-Zero Gold protocol (Illumina) as described (22) . Total RNAs were depleted for ribosomal RNA, fragmented, and cDNA was synthesized using SuperScript III Reverse Transcriptase (Thermo Fisher Scientific). cDNA was subjected to AMPure beads (Beckman Coulter) and adenylated to prime for adapter ligation. DNA fragments were amplified using PCR with the following settings: 30 s, 98°C; (10 s, 98°C; 30 s, 60°C; 30 s, 72°C) × 9-13 cycles; 5 min, 72°C, followed by a final cleanup. The cycle number was set to prevent saturation and overamplification of individual samples. Libraries were quality-controlled using a Bioanalyzer instrument (Agilent Technologies). Libraries were subjected to 100-bp single-end sequencing on the X Ten platform (Illumina) at the Beijing Genomics Institute (BGI; Hong Kong, China).