GSM5560981: HSD2; Mus musculus; RNA-Seq
After the liver is taken out of the living body, rinse with 0.9% saline immedietely to remove blood stains and dirt, remove connective tissue and adipose tissue and other tissue types that are not required for research, and absorb the liquid on the surface of the material; divide the tissue sample with a clean blade into a small piece of about 50 mg (about the size of a soybean), after quick freezing in liquid nitrogen, put it into a clean preservation tube with a screw cap. Storing in a refrigerator at minus eighty degrees and shipping on dry ice. And RNA was harvested using Trizol reagent.Total RNA was extracted from the tissues using Trizol(Invitrogen, Carlsbad, CA, USA) accordingto manual instruction. About 60 mg of tissues were ground into powder by liquid nitrogen in a 2mL tube, followed by being homogenized for 2 minutes and rested horizontally for 5 minutes. Themix was centrifuged for 5 minutes at 12,000×g at 4°C, then the supernatant was transferred into anew EP tube with 0.3 mL chloroform/isoamyl alcohol (24:1). The mix was shacked vigorously for15s, and then centrifuged at 12,000×g for 10 minutes at 4°C. After centrifugation, the upperaqueous phase where RNA remained was transferred into a new tube with equal volume ofsupernatant of isopropyl alcohol, then centrifuged at 13,600 rpm for 20 minutes at 4°C. Afterdeserting the supernatant, the RNA pellet was washed twice with 1 mL75% ethanol, then the mixwas centrifuged at 13,600 rpm for 3 minutes at 4°C to collect residual ethanol, followed by thepellet air dry for 5-10 minutes in the biosafety cabinet. Finally, 25µL~100µL of DEPC-treatedwater was added to dissolve the RNA. Subsequently, total RNA was qualified and quantified usinga Nano Drop and Agilent 2100 bioanalyzer (Thermo Fisher Scientific, MA, USA). Oligo(dT)-attached magnetic beads were used to purified mRNA. Purified mRNA was fragmentedinto small pieces with fragment buffer at appropriate temperature. Then First-strand cDNA wasgenerated using random hexamer-primed reverse transcription, followed by a second-strand cDNAsynthesis. afterwards, A-Tailing Mix and RNA Index Adapters were addedby incubating to endrepair. The cDNA fragments obtained from previous step were amplified by PCR, and productswere purified by Ampure XP Beads, then dissolved in EB solution. The product was validated onthe Agilent Technologies 2100 bioanalyzer for quality control. The double stranded PCR productsfrom previous step were heated denatured and circularized by the splint oligo sequence to get thefinal library. The single strand circle DNA (ssCir DNA) was formatted as the final library. Thefinal library was amplified with phi29 to make DNA nanoball (DNB) which had more than 300copies of one molecular, DNBs were loaded into the patterned nanoarray andsingleend50basesreads were generated on BGIseq500 platform (BGI-Shenzhen, China).