GSM9141093: GSC3028 cells,shCONT-3; Homo sapiens; RNA-Seq
Add the equal volume of 70% ethanol to the collection tube, mix by pipetting.Transfer 700 μL mixed solution, to a RNeasy spin column placed in a 2 mL collection tube.Centrifuge at 10,000×g for 30 s and allow it to empty by gravity flow.Add 700 μL Buffer RW1 to RNeasy spin column. Centrifuge at 10,000×g for 30 s and discard the filtrate.Add 500 μL Buffer RPE to RNeasy spin column. Centrifuge at 10,000×g for 30 s and discard the filtrate.Add 500 μL Buffer RPE to RNeasy spin column. Centrifuge at 10,000×g for 30 s and discard the filtrate.Centrifuge at 16,000×g for 2 min and transfer the RNeasy spin column to the 1.5 mL centrifuge tube. Add 35 μL RNase-free water to RNeasy spin column. Stay at room temperature for 1min.Add 35 μL RNase-free water to RNeasy spin column. Stay at room temperature for 1min.Centrifuge at 16,000×g for 2 min at room temperature to collect RNA. RNA-Seq (Quantification) uses a new generation of high-throughput sequencing technology to study the gene expression pattern of eukaryotic cells and provide accurate digital expression profile detection by sequencing and comparing transcripts. Transcriptome research is the basis and starting point of gene function and structure research. Through the new generation of high-throughput sequencing, almost all transcripts of a specific tissue or organ of a species can be obtained comprehensively and quickly. 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. 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).