GSM8262893: tissue, MD_2, Disease group and AVFP therapy; Mus musculus; RNA-Seq
Appropriate amount of tissue samples were taken, 1.5mL of TRIzol lysate was added, and put into a tissue grinder for 30s grinding, and then removed and left for 5min to fully lysate the tissue cells.The ground and crushed tissue samples were centrifuged, and the supernatant was transferred into an EP tube filled with 300µL chloroform/isoamyl alcohol (24:1). The supernatant was absorbed into a new 1.5 mL centrifuge tube, and dissolved and precipitated with 20-200 µL DEPC water or RNase-free water. Select the corresponding testing methods for quality inspection according to the requirements of samples and products.A certain amount of RNA samples are denatured at suitable temperature to open their secondary structure, and mRNA is enriched by oligo (dT) -attached magnetic beads.The reaction system is configured. After reacting at the suitable temperature for a fixed period of time, RNAs are fragmented.Prepare the first-strand synthesis reaction system, and set up the reaction program, synthesize the first- strand cDNA, prepare the second-strand synthesis reaction system, and set up the reaction program to synthesize the second-strand cDNA.After the reaction system and program are configured and set up, double-stranded cDNA fragments are subjected to end-repair, and then a single 'A' nucleotide is added to the 3' ends of the blunt fragments. The reaction system and program for adaptor ligation are subsequently configured and set up to ligate adaptors with the cDNAs.The PCR reaction system and program are configured and set up to amplify the product.The corresponding library quality control protocol will be selected depending upon product requirements.Single-stranded PCR products are produced via denaturation. The reaction system and program for circularization are subsequently configured and set up. Single-stranded cyclized products are produced, while uncyclized linear DNA molecules are digested.