title: GSM5369073: L4_RNA-seq; Mus musculus; RNA-Seq
library_construction_protocol: Total RNA was extracted from C2C12 myotubes using easy-BLUETM total RNA extraction kit according to the manufacturer's instructions (iNtRON). RNA quality was assessed by Agilent 2100 bioanalyzer (Agilent Technologies, Amstelveen, The Netherlands), and RNA quantification was performed using ND-2000 Spectrophotometer (Thermo Inc., DE, USA). Libraries were prepared from total RNA using the NEBNext Ultra II Directional RNA-Seq Kit (NEW ENGLAND BioLabs, Inc., UK). The isolation of mRNA was performed using the Poly(A) RNA Selection Kit (LEXOGEN, Inc., Austria). The isolated mRNAs were used for the cDNA synthesis and shearing, following manufacture's instruction. Indexing was performed using the Illumina indexes 1- 12. The enrichment step was carried out using of PCR. Subsequently, libraries were checked using the Agilent 2100 bioanalyzer (DNA High Sensitivity Kit) to evaluate the mean fragment size. Quantification was performed using the library quantification kit using a StepOne Real-Time PCR System (Life Technologies, Inc., USA). High-throughput sequencing was performed paired-end reads (2 × 100 bp) using HiSeq 2500 (Illumina, Inc., USA).