peripheral blood 38
Taking advantage of the structural feature that most mRNAs of eukaryotes carry a polyA tail, mRNAs with polyA tails are enriched using Oligo(dT) magnetic beads. The purified mRNA is fragmented into small segments using fragmentation buffer at an appropriate temperature. Random hexamer primers are used for reverse transcription to generate the first-strand cDNA. Synthesize the second-strand cDNA (for strand-specific libraries: dUTP is used instead of dTTP during the second-strand synthesis, so dUTP is incorporated into the second strand of cDNA; the high-fidelity DNA polymerase used in this method cannot amplify DNA templates containing uracil, thus achieving strand specificity), while completing end repair and dA-Tailing. Perform Adapter Ligation; after ligation, conduct DNA magnetic bead purification and size selection to obtain a library with insert fragments of 250-350bp. Amplify the ligation product via PCR, then purify the amplified product using DNA magnetic beads again, and dissolve the product in Nuclease-Free Water (note: "Nuclear Free Water" in the original text is a common typo, and the standard term is "Nuclease-Free Water"). After library construction is completed, first use a Qubit Fluorometer for concentration detection, and then use a Qsep437 High-Throughput Biological Fragment Analyzer for fragment size detection.