US 12,435,333 B2
Parallel analysis of RNA 5′ ends from low-input RNA
Michael Douglas Nodine, Vienna (AT); Michael Schon, Vienna (AT); and Max Kellner, Cambridge, MA (US)
Assigned to GMI—Gregor-Mendel-Institut fuer Molekulare Pflanzenbiologie GmbH, Vienna (AT)
Appl. No. 17/264,467
Filed by GMI—Gregor-Mendel-Institut fuer Molekulare Pflanzenbiologie GmbH, Vienna (AT)
PCT Filed Jul. 30, 2019, PCT No. PCT/EP2019/070458
§ 371(c)(1), (2) Date Jan. 29, 2021,
PCT Pub. No. WO2020/025599, PCT Pub. Date Feb. 6, 2020.
Claims priority of provisional application 62/712,156, filed on Jul. 30, 2018.
Prior Publication US 2021/0284997 A1, Sep. 16, 2021
Int. Cl. C12Q 1/68 (2018.01); C12N 15/10 (2006.01); C12Q 1/6806 (2018.01); C12Q 1/6874 (2018.01)
CPC C12N 15/1096 (2013.01) [C12Q 1/6806 (2013.01); C12Q 1/6874 (2013.01)] 16 Claims
 
1. A method for generating multiple sequencing libraries from a single full-length cDNA library comprising:
a) providing RNA molecules comprising RNA 5′ ends and reverse transcribing the RNA molecules with at least one template switching oligonucleotide (TSO) to generate the single full-length cDNA library, wherein cDNA in the single full-length cDNA library comprise sequences of the TSO at the 5′-end of the cDNA,
b) isometric PCR amplification of the cDNA from said single full-length cDNA library into double-stranded (ds) cDNA,
c) fragmenting said ds cDNA to generate ds cDNA fragments,
d) adding bridge PCR compatible forward and reverse adapters to said ds cDNA fragments to generate a pool of ds cDNA fragments comprising sequences of the forward and reverse adapters,
e) wherein c) and d) are optionally performed simultaneously by tagmentation of the ds cDNA fragments, and
f) generating the multiple sequencing libraries by separating the pool of ds cDNA fragments into a first and a second aliquot wherein
(i) the first aliquot is amplified using primers complementary to the sequences of the forward and reverse adapters added in d) to generate a sequencing compatible gene body cDNA library and
(ii) the second aliquot is further split into a first and a second sub aliquot, wherein
the first sub aliquot is amplified using a primer matching the sequence of the at least one TSO in a) and a primer matching the sequence of the forward adapter added in d) and
the second sub-aliquot is amplified using a primer matching the sequence of the at least one TSO in a) and a primer matching the sequence of the reverse adapter added in d), and
generating, from the first and second sub-aliquots, a sequencing compatible 5′ end library, wherein a) to f) are performed sequentially.