GSM8886438: 1-2; Candidozyma auris; RNA-Seq
Following sample collection, 400 μL of ice-cold lysis buffer (composed of 0.1 M LiCl, 0.1 M Tris [pH 7.5], 5% SDS, and 2% β-mercaptoethanol) was added to resuspend the samples. Subsequently, 200 μL of acid-washed glass beads and 200 μL of PCIA (pH 4.5, P120617, Aladdin) were incorporated into the mixture. This combination was vortexed at maximum speed at 40C for 5 minutes, then placed on ice for an additional 5 minutes. Following this step, the mixture was centrifuged at maximum speed at 40C for 10 minutes, and the supernatant was carefully collected. To purify the supernatant, it was sequentially extracted with 200 μL of PCIA and then with 300 μL of chloroform. After purification, 1 mL of 100% ethanol was added to precipitate the RNA, which was then incubated at -800C for 30 minutes. The mixture was subsequently centrifuged at maximum speed at 40C for 10 minutes, and the resulting pellet was washed once with 75% ethanol. Finally, the pellet was dissolved in 40 μL of nuclease-free water. 1. Take a certain amount of total RNA samples, and use oligo dT beads to enrich mRNA with poly A tail; 2. mRNA molecules were fragmented into small pieces; 3. The fragmented mRNA was synthesized into first strand cDNA using random primers;  4. The second strand cDNA was synthesized with dUTP instead of dTTP; 5. The synthesized cDNA was subjected to end-repair and 3' adenylated. Adaptors were ligated to the ends of these 3' adenylated cDNA fragments; 6. Digest the U-labeled second-strand template with Uracil-DNA-Glycosylase (UDG) and perform PCR amplification; 7. Library quality control; 8. Library circularization;