IP Library › Granted Patent US 12,545,950
Granted Patent B2
US 12,545,950 · App. 17/311,521 · Granted Feb 10, 2026

Methods for preparing CDNA samples for RNA sequencing, and CDNA samples and uses thereof

Inventors: Yexun Wang (Ellicott City, MD); Marcus Lewis (Clarksburg, MD)
Assignee: QIAGEN Sciences, LLC
C12Q1/6853C12Q1/6848
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Quick Facts
Patent No.
US 12,545,950
App. No.
17/311,521
Granted
Feb 10, 2026
Kind
B2
Abstract

The invention relates to methods for preparing cDNA samples for RNA sequencing using random priming oligonucleotides comprising a cell barcode (cID), a unique molecular index (UMI), and a random sequence region, and performing a reverse transcription reaction (RT). The invention also relates to cDNA samples prepared by the methods and uses thereof.

Claims (11)

1 . A method of generating a cDNA sample, comprising

adding to an RNA sample, a plurality of random priming oligonucleotides, each random priming oligonucleotide comprising a cell barcode (cID), a unique molecular index (UMI), and a random sequence region of 5 to 8 nucleotides, and

performing a reverse transcription reaction (RT),

wherein the random sequence region consists of (a) random nucleotides selected from A, C, and T bases; (b) 5 random nucleotides selected from A, C, G, and T bases and 3 G bases; (c) 5 random nucleotides selected from A, C, G, and T bases and 3 G bases in a 5′ to 3′ direction; (d) 5 random nucleotides selected from A, C, G, and T bases and 3 T bases; or (e) 5 random nucleotides selected from A, C, G, and T bases and 3 T bases in a 5′ to 3′ direction.

2 . The method of claim 1 , wherein the random priming oligonucleotides further comprise a universal PCR handle.

3 . The method of claim 2 , further comprising adding a template switching oligonucleotide (TSO) comprising a 5′ universal PCR handle and performing a template switching reaction (TS) to generate a cDNA sample comprising a universal PCR handle at the 5′ end and at the 3′ end.

4 . The method of claim 3 , wherein the TS is decoupled from the RT.

5 . The method of claim 2 , further comprising adding a homopolymer oligonucleotide tail to a first strand cDNA by adding a terminal transferase (TdT) and then performing a primer extension reaction using a template switching oligonucleotide (TSO) comprising a 5′ universal PCR handle, wherein the TSO hybridizes to the homopolymer oligonucleotide tail and the primer extension reaction generates a second strand cDNA comprising a universal PCR handle at the 5′ end and at the 3′ end.

6 . The method of claim 1 , wherein two or more of the random priming oligonucleotides bind to a single RNA molecule.

7 . The method of claim 1 , further comprising performing an amplification reaction to generate a cDNA sample.

8 . The method of claim 7 , further comprising sequencing the cDNA sample.

Assignments (1)
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Dec 14, 2021
From: WANG, YEXUN; LEWIS, MARCUS
To: QIAGEN SCIENCES, LLC
Reel/Frame 058390/0747 →
Continuity (2)
Provisional Application 62776697 · Dec 7, 2018
Related Publication 20220017954A1 · Jan 20, 2022
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