GSM9421682: SFSN-Neu_1; Rattus norvegicus; RNA-Seq
1. Introduction This protocol allows to extract both high-quality genomic DNA and total RNA from the same sample. The purified DNA and RNA can be used directly for library preparation. 2.Experimental Procedures 2.1 Sample Lysis 2.1.1 Transfer 30mg of tissue into a 2mL tube with beads. 2.2.1 Add 600µL Buffer RLT Plus. If using snap-frozen cells (no more than 1 × 10⁷ cells), simply add 600µL Buffer RLT Plus directly into the same tube. Homogenize at 65Hz for 60s, then stay at room temperature for 5min. 2.2.3 Centrifuge at 12,000g for 3min. Transfer the supernatant to an AllPrep DNA spin column in a collection tube. Centrifuge at 10,000g for 30s in room temperature. 2.2.4 Collect the flow-through for RNA purification. 2.2 RNA Purification 2.2.1 Add one volume of 70% ethanol to the flow-through. Mix by pipetting at least 5 times. 2.2.2 Transfer 700µL of the mixture into an RNeasy spin column in a collection tube. Centrifuge at 10,000g for 30s; discard the flow-through. Repeat until all mixture has passed through the column. 2.2.3 Add 700μL Buffer RW1 to RNeasy spin column, centrifuge at 10,000×g for 30s in room temperature and discard the filtrate. 2.2.4 Add 500μL Buffer RPE to RNeasy spin column, centrifuge at 10,000×g for 30s in room temperature and discard the filtrate. 2.2.5 Add 500μL Buffer RPE to RNeasy spin column, centrifuge at 10,000×g for 30s in room temperature and discard the filtrate. 2.2.6 Centrifuge at 16,000g for 2min to dry the purification column. Transfer the RNeasy spin column to a 1.5mL tube. 2.2.7 Add 30µL RNase-free water to the RNeasy spin column. Stay at room temperature for 1min, then centrifuge at 16,000g for 2min to elute RNA. 2.2.8 Check RNA quality as required. 2.3 DNA Purification 2.3.1 Add 500μL Buffer AW1 to AlIPrep DNA spin column, centrifuge at 10,000×g for 30s in room temperature and discard the filtrate. 2.3.2 Add 500μL Buffer AW2 to AlIPrep DNA spin column, centrifuge at 10,000×g for 30s in room temperature and discard the filtrate. 2.3.3 Centrifuge at 16,000g for 2min to dry the purification column. Transfer the AllPrep DNA spin column to a 1.5mL tube. 2.3.4 Add 30µL Buffer EB to theAllPrep DNA spin column. Stay at room temperature for 1min, then centrifuge at 16,000g for 2min to elute DNA. 2.3.5 Check DNA quality as required. 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).