GSM4848134: CKD1; Mus musculus; miRNA-Seq
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. Four samples were pooled into one sample for miRNA sequencing at each time-point. Subsequently, total RNA was qualified and quantified using a Nano Drop and Agilent 2100 bioanalyzer (Thermo Fisher Scientific, MA, USA). Total RNA was separated by 15% agarose gels to extract the small RNA (18-30 nt). After precipitated by ethanol and centrifugal enrichment of small RNA sample, the library was prepared according to the method and process of Small RNA Sample Preparation Kit (Illumina, RS-200-0048). The main contents are as follows: 1) Connecting the 3' adaptor to the separated small RNA. 2) Connecting the 5' adaptor to the separated small RNA. 3) RT－PCR. 4) Recycling strips of 145-160bp (22-30nt RNA).