GSM7833044: CON6; Homo sapiens; RNA-Seq
1. Introduction This protocol applies to the isolation and purification of RNA from whole blood that are stabilized on PAXgene Blood RNA Tubes to provide high-quality RNA for library preparation. 2. Experimental Procedures 2.1 Remove the blood sample from the refrigerator and balance at room temperature for 2 h. 2.2 Centrifuge at 3000~5000×g for 10 min, discard the supernatant. 2.3 Add 4 mL RNase-free water, vortex shaking until the cells are dispersed, centrifugation at 3000-5000×g for 10 min and removal of the supernatant. 2.4 Add 350 μL BR1, vortex and shake well, add 300 μL BR2 and 40 μL proteinase K, vortex and shake well for 5 s and incubate at 55℃, 400~1400 rpm for 10 min.  2.5 Let the mixture pass through the PAXgene Shredder spin column, centrifuge at full speed for 3 min, and then transfer the supernatant to a 1.5 mL centrifuge tube, add 350 μL ethanol absolute and mix thoroughly by vortexing. 2.6 Transfer the supernatant to PAXgene RNA spin column, centrifuge at 8000~20000×g for 1 min. 2.7 Add 350 μL BR3, centrifuge at 8000~20000×g for 1 min. 2.8 Add 70 μL DNA digestion buffer and 10μL DNase I, stay at 20-30℃ for 15 min. 2.9 Add 350 μL BR3, centrifuge at 8000~20000×g for 1 min, discard the filtrate. 2.10 Add 500 μL BR4, centrifuge at 8000~20000×g for 1 min, discard the filtrate. 2.11 Add 500 μL BR4, centrifuge at 8000~20000×g for 3 min, discard the filtrate. 2.12 Transfer the column into a new 1.5 mL tube, add 20-50 μL BR5, centrifuge at 8000~20000×g for 1min to elute the DNA. 1. Introduction 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. 2.Experimental Procedure 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).