GSM5115353: small RNA control_3; Rattus norvegicus; ncRNA-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 4Â°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 4Â°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 4Â°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 25ÂµL-100ÂµL 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). Library was prepared with 1 µg total RNA for each sample. Total RNA was purified by electrophoretic separation on a 15% urea denaturing polyacrylamide gel electrophoresis (PAGE) gel and small RNA regions corresponding to the 18-30 nt bands in the marker lane (14-30 ssRNA Ladder Marker, TAKARA) were excised and recovered. Then the 18-30 nt small RNAs were ligated to adenylated 3' adapters annealed to unique molecular identifiers (UMI), followed by the ligation of 5' adapters. The adapter-ligated small RNAs were subsequently transcribed into cDNA by Superscript II Reverse Transcriptase (Invitrogen, USA) and then several rounds of PCR amplification with PCR Primer Cocktail and PCR Mix were performed to enrich the cDNA fragments. The PCR products were selected by agarose gel electrophoresis with target fragments 110~130 bp, and then purified by QIAquick Gel Extraction Kit (QIAGEN, Valencia, CA). The 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 final ligation PCR products were sequenced using the DNBSEQ platform (BGI- Shenzhen, China).