Dolang Sheep (D)
ONT sequencing is a new generation sequencing technology based on nanopore single-molecule real-time electrical signal, the sequencing principle of each platform is the same. Under the guidance of motor proteins, double-stranded DNA/RNA can bind to nanopore proteins that are inlaid on biofilm and then get unpackaged. Under the action of voltage difference on both sides of the biofilm, DNA/RNA strand passes through the nanopore channel protein at a certain rate. Due to the difference in the chemical properties of different bases on the DNA/RNA strand, when a single base or DNA molecule passes through the nanopore channel, different electrical signals will be generated. By detecting and classifying these signals, the corresponding base type can be calculated and the real-time determination of the sequence can be completed.Experiment workflow will be performed according to standard protocol provided by Oxford Nanopore Technologies(ONT), including sample QC, library construction, library QC, library sequencing and so on, mainly including the following steps:1. RNA was extracted and the purity, concentration and integrity of RNA samples were detected by advanced equipment to ensure that qualified samples can be used for transcriptome sequencing.2. Library construction:1) primer annealing, reverse transcription to get cDNA, add switch oligo;2) synthesis of complementary chains;3) DNA damage repair and end repair, magnetic beads purification.3. Add the sequencing adaptors, sequence samples on the machine.