GSM5115329: control_1; Rattus norvegicus; RNA-Seq
Total RNA was extracted from the Sertoli cells using Trizol (Invitrogen, Carllsbad, CA,   USA ) according to the manual instructions .The mix was centrifuge at 12000xg 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 12000xg for 10min at 4°C, the aqueous phase was transferred to a new   1.5 ml tube which was add equal volume of supernatant of isopropyl alcohol. The mix   was centrifuged at 12000 xg 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 (NanoDrop, Madison, USA)  and Agilent 2100 bioanalyzer (Agilent, Santa Clara, USA). Take 200ng total RNA samples (only for human or mouse) and use RNase H to remove the rRNA.   DNase I digested double-stranded and single-stranded DNA presenting in RNA samples. mRNA   molecules were fragmented into small pieces using fragmentation reagent. First-strand cDNA was   generated by First Strand Master Mix and Super Script II reverse transcription, then add Second   Strand Master Mix to synthesize the second-strand cDNA. Purify the DNA with Ampure XP Beads,   then carry out end repair. The end-repaired products were purified by magnetic beads and re-dissolved in EB Solution. The purified fragmented cDNA was combined with A-Tailing Mix, and   incubated. Then combine the Adenylate 3'Ends DNA, RNA Index Adapter and Ligation Mix,   incubate. Purify the product DNA with Ampure XP Beads. Several rounds of PCR amplification   with PCR Primer Cocktail and PCR Master Mix were performed to enrich the cDNA fragments.   Then the PCR products were purified with Ampure XP Beads. The libraries were assessed quality   and quantity 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 amplified on cBot to generate the cluster on the flowcell. And the   amplified flowcell will be sequenced single end on the HiSeq X-ten paltform (BGI-Shenzhen,  China).