GSM9192999: Adipose tissue,WT3; Mus musculus; RNA-Seq
The RNA of the total sample was separated and purified using the TransZol Up Plus RNA Kit(Full Gold) according to the operation plan provided by the manufacturer. Then, the quantity and purity of total RNA were controlled using Pono-650 (Porabio). The integrity of RNA was then detected by Qsep100 (BiOptic, Taiwan). The concentration >20ng/μL, the RIN value >7.0, and the total RNA>1μg meet the requirements for downstream experiments. The PolyA-containing mRNA was specifically captured using oligo(dT) magnetic beads (item No. N403, Vazyme Novozyme) through two rounds of purification. The captured mRNA was fragmented under high-temperature conditions using magnesium ion disruption (all the following are from the same kit, item number NR605, Vazyme Novozyme). cDNA is synthesized from fragmented RNA under the action of reverse transcriptase. Then, two-strand synthesis is carried out, and dUTP is simultaneously incorporated into the two strands to complete the ends of the double-stranded DNA to flat ends. Then add an A base to each end of it to enable it to connect with the connector with a T base at the end, and then use magnetic beads to screen and purify the size of its fragments. Then, through PCR - pre-denaturation at 98 ° C for 45 seconds, denaturation at 98 ° C for 15 seconds, denaturation at 60 ° C for 30 seconds, and denaturation at 72 ° C for 30 seconds, a total of 12 cycles were carried out. Finally, it was extended to 72 ° C for 1 minute to form a library with fragment sizes of 370-470bp.