title: GSM5468096: C1_Male_D3_rep2; Mus musculus; RNA-Seq
library_construction_protocol: Total RNA was extracted from the tissues using Trizol (Invitrogen, Carlsbad, CA, USA) according to the manual instructions. For tissue samples, grind about 60mg with liquid nitrogen into powder and transfer the powder samples into the 2ml tube contains 1.5ml Trizol reagent. The mix was centrifuge at 12000×g for 5min at 4°C. The supernatant was transferred to a new 2.0ml tube which was added 0.3ml of Chloroform/isoamyl alcohol (24:1) per 1.5ml of Trizol reagent. After the mix was centrifuged at 12000×g for 10min at 4°C, the aqueous phase was transferred to a new 1.5mL tube which was add equal volume of supernatant of isopropyl alcohol. The mix was centrifuged at 12000×g for 20min at 4°C and then removed the supernatant. After washed with 1ml 75% ethanol, the RNA pellet was air-dried in the biosafety cabinet and then dissolved by add 25µL~100µL of DEPC-treated water. Subsequently, total RNA was qualified and quantified using a Nano Drop and Agilent 2100 bioanalyzer (Thermo Fisher Scientific, MA, USA). Approximately 1µg total RNA per sample was treated with Ribo-Zero™ Magnetic Kit(Epicentre) to deplete rRNA. The retrieved RNA was fragmented by adding First Strand Master Mix(Invitrogen). First-strand cDNA was generated using random primers reverse transcription, followed by a second-strand cDNA synthesis. The synthesized cDNA was subjected to end-repair and then was 3' adenylated. Adapters were ligated to the ends of these 3' adenylated cDNA fragments. Several rounds of PCR amplification with PCR Primer Cocktail and PCR Master Mix are performed to enrich the cDNA fragments. Then the PCR products are purified with Ampure XP Beads. The final library was quality and quantitated in two methods: check the distribution of the fragments size using the Agilent 2100 bioanalyzer, and quantify the library using real-time quantitative PCR (QPCR) (TaqMan Probe). The Qualified libraries were sequenced pair end on the Hiseq X-ten platform (BGI-Shenzhen, China).