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METHOD FOR GENERATING A RNA-SEQUENCING LIBRARY

  • US 20180230527A1
  • Filed: 08/24/2016
  • Published: 08/16/2018
  • Est. Priority Date: 08/24/2015
  • Status: Active Grant
First Claim
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1. A method of RNA sequencing, comprising:

  • (i) providing RNA;

    (ii) generating a single-stranded first DNA strand, which is complementary to the RNA of (i), by subjecting the RNA of (i) to reverse transcription by using a reverse transcriptase and a first set of oligonucleotide primers;

    (iii) generating a double-stranded second DNA strand using a DNA polymerase, a second set of oligonucleotide primers, and the single-stranded first DNA generated in (ii);

    (iv) ligating adapters to the double-stranded second DNA of (iii) to obtain a generated DNA; and

    (v) sequencing the generated DNA;

    wherein;

    a) the first set of oligonucleotide primers comprises a moiety at its/their 5′

    terminal nucleotide, which blocks ligation at a 5′

    terminus of the single-stranded first DNA strand generated in (ii);

    orb) the second set of oligonucleotide primers comprises a moiety at its/their 5′

    terminal nucleotide, which blocks ligation at a 5′

    terminus of the double-stranded second DNA strand generated in (iii).

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