title: Illumina HiSeq 2500 sequencing; Expression profiling of 3 and 24 months mouse skeletal muscle (RNA-seq)
design: Expression profiling of 3 and 24 months mouse skeletal muscle (RNA-seq)
library_construction_protocol: The wild-type male mice were purchased from National Laboratory Animal Center and maintained in a specific pathogen-free facility. Skeletal muscle tissues were obtained from the wild-type mice at 3 months and 24 months of age. The animal protocol was approved by the Institutional Animal Care and Use Committee of National Yang-Ming University. Total RNA was isolated from mouse skeletal muscle using TRIzol Reagent (Life Technology). Total RNA was extracted twice with phenol-chloroform and precipitated with isopropanol. Subsequently, in order to remove the chemicals, total RNA was precipitated with ethanol twice and dissolved in autoclaved water. The sequencing library for mRNA-seq was prepared using TruSeq RNA Sample Preparation Kit v2 (Illumina) according to manufacturer's instruction. Briefly, 4 mg of total high quality RNA (RNA integrity number > 8) was subjected to poly-A mRNA isolation using poly-T oligonucleotides. The poly-A mRNA was fragmented and the first-strand cDNA was synthesized using random hexamers followed by second-strand cDNA synthesis, end repair, addition of a single A base and adapter ligation. The adapter-ligated double-stranded cDNA library was enriched with 7-9 cycles of PCR using KAPA HiFi DNA polymerase (Kapa Biosystems).
attributes.tag: Experimental Factor: 3
attributes.value: age