title: RNA-Seq of Escherichia coli: Escherichia coli Nissle 1917
design: Sample QCSelect the corresponding testing methods for quality inspection according to the requirements of samples and products.mRNA IsolationA 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.mRNA FragmentationThe reaction system is configured. After reacting at the suitable temperature for a fixed period of time, RNAs are fragmented.cDNA SynthesisPrepare 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.End Repair, Add A and Adaptor LigationAfter 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.PCRThe PCR reaction system and program are configured and set up to amplify the product.Library QCThe corresponding library quality control protocol will be selected depending upon product requirements.CircularizationSingle-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.SequencingSingle-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).