GSM7664324: Ctrl; Staphylococcus aureus; RNA-Seq
2.1 Incubate 10% SDS at 37℃，add 60-100 µL elution buffer for each sample in 2.0 mL tube and incubate at 95℃. 2.2 Add 500 µL ice-cold Zirconia Beads to 2.0 mL screwed tube，add 480 µL Phenol/chloroform/iso-amyl alcohol 25:24:1）、480 µL Lysis Buffer and 48 µL 10% SDS. 2.3 Transfer appropriate microbial sample to 2.0 mL screwed tube, homogenize at 65Hz for 60 s，stay at room temperature for 5 min. 2.4 Centrifuge at 15,000×g for 5 min at room temperature. 2.5 Transfer supernatant to new 5.0 mL tube，add 1.9 mL Binding Buffer、1.2 mL ethanol absolute and mix thoroughly. 2.6 Transfer 700 µL mixed solution to Filter Cartridge with collection tube，centrifuge for 15,000×g for 30 s at room temperature and discard the filtrate. 2.7 Repeat 2.6，until all mixed solution pass the Filter Cartridge. 2.8 Add 700 µL Wash Solution 1，centrifuge at 10,000×g for 30 s at room temperature and discard the filtrate. 2.9 Add 500 µL Wash Solution 2/3，centrifuge at 10,000×g for 30 s at room temperature and discard the filtrate. 2.10 加入500 µL Wash Solution 2/3，centrifuge at 10,000×g for 30 s at room temperature and discard the filtrate. 2.11 Centrifuge at 15,000×g for 2 min at room temperature, transfer the Filter Cartridge to a new 2.0 mL collection tube. 2.12 Add 60-100 µL prewarmed elution buffer to Filter Cartridge and stay for 2 min，centrifuge at 15,000×g for 1min and collect the RNA solution. 2.1 Sample QC Select the corresponding testing methods for quality inspection according to the requirements of samples and products. 2.2 mRNA Isolation 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. 2.3 mRNA Fragmentation The reaction system is configured. After reacting at the suitable temperature for a fixed period of time, RNAs are fragmented. 2.4 cDNA Synthesis 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. 2.5 End Repair, Add A and Adaptor Ligation 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. 2.6 PCR The PCR reaction system and program are configured and set up to amplify the product. 2.7 Library QC The corresponding library quality control protocol will be selected depending upon product requirements. 2.8 Circularization 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. 2.9 Sequencing Single-stranded circle DNA molecules are replicated via rolling cycle amplification, and a DNA nanoball (DNB) which contain multiple copies of DNA is generated. Sufficient quality DNBs are then loaded into patterned nanoarrays using high-intensity DNA nanochip technique and sequenced through combinatorial Probe-Anchor Synthesis (cPAS).