US 12,319,910 B2
Second strand direct
Ronald Lebofsky, Kensington, CA (US); and Jeremy Agresti, Richmond, CA (US)
Assigned to Bio-Rad Laboratories, Inc., Hercules, CA (US)
Filed by Bio-Rad Laboratories, Inc., Hercules, CA (US)
Filed on Jun. 27, 2023, as Appl. No. 18/214,941.
Application 18/214,941 is a continuation of application No. 17/102,111, filed on Nov. 23, 2020, granted, now 11,725,206.
Application 17/102,111 is a continuation of application No. 16/687,411, filed on Nov. 18, 2019, granted, now 10,876,112, issued on Dec. 29, 2020.
Application 16/687,411 is a continuation of application No. 15/668,321, filed on Aug. 3, 2017, granted, now 10,676,736, issued on Jun. 9, 2020.
Claims priority of provisional application 62/522,232, filed on Jun. 20, 2017.
Claims priority of provisional application 62/371,638, filed on Aug. 5, 2016.
Prior Publication US 2024/0076658 A1, Mar. 7, 2024
This patent is subject to a terminal disclaimer.
Int. Cl. C12N 15/10 (2006.01)
CPC C12N 15/1096 (2013.01) [C12N 15/1065 (2013.01)] 18 Claims
 
1. A method for tagging a cDNA polynucleotide, the method comprising:
providing a double-stranded mRNA: cDNA hybrid comprising a first strand cDNA polynucleotide having a 5′ end and a 3′ end and hybridized to a complementary mRNA having a 5′ end and a 3′ end, wherein the 5′ end of the first strand cDNA polynucleotide comprises a first adapter sequence or complement thereof;
synthesizing one or more second strand cDNA polynucleotides that are complementary to and hybridized to the first strand cDNA polynucleotide, wherein the synthesizing comprises:
i) hybridizing random primers to the first strand cDNA of the mRNA: cDNA hybrid and extending hybridized random primers to form the one or more second strand cDNA polynucleotides;
contacting the double-stranded cDNA polynucleotide with an adapter-loaded tagmentase, thereby forming a reaction mixture comprising a tagged double-stranded cDNA polynucleotide comprising a first end and a second end, wherein the first end comprises the first adapter sequence and complement thereof, and the second end comprises a second adapter sequence and complement thereof,
forming partitions comprising (a) amplification primers comprising at least one primer comprising a partition-specific barcode and (b) the first strand cDNA and the second strand cDNAs and
amplifying the first and second strand cDNAs with the amplification primers.