Methods and kits for depleting undesired nucleic acids

A method for depleting at least one undesired RNA species having a 5′ end of the undesired RNA and a 3′ end of the undesired RNA, comprising:

• combining a sample with at least one single-stranded nucleic acid blocker having a 5′ end of the blocker and a 3′ end of the blocker, at least one 3′ adapter, and at least one first ligase to form a first reaction composition and incubating under conditions suitable for first ligation products to be formed, wherein the single-stranded nucleic acid blocker is added before, simultaneously with, or after the 3′ adapter, and wherein the first reaction composition is incubated under conditions suitable for the single-stranded nucleic acid blocker to anneal with the undesired RNA to form an RNA-blocker duplex, wherein the RNA-blocker duplex has a blunt duplex end comprising the 5′ end of the undesired RNA and the 3′ end of the blocker, or wherein the blocker extends beyond the 5′ end of the undesired RNA in the RNA-blocker duplex, wherein the RNA-blocker duplex is formed before, simultaneously with, or after the first ligation products are formed, and wherein the blocker does not form a unimolecular duplex under the conditions suitable for the single-stranded nucleic acid blocker to anneal with the undesired RNA to form an RNA-blocker duplex;
• separating the first ligation products from the first reaction composition;
• combining the separated first ligation products with at least one 5′ adapter and at least one second ligase to form a second reaction composition and incubating under conditions suitable for second ligation products to be formed;
• wherein neither the 5′ end of the blocker nor the 3′ end of the blocker is ligated to the undesired RNA;
• and
• amplifying the second ligation products to generate a pool of amplification products, wherein amplification products corresponding to the at least one undesired RNA species are depleted.

A method for depleting at least one undesired RNA species having a 5′ end of the undesired RNA and a 3′ end of the undesired RNA, comprising:

• combining a sample with at least one 3′ adapter and at least one first ligase to form a first reaction composition and incubating under conditions suitable for first ligation products to be formed;
• combining the first reaction composition comprising the first ligation products with at least one 5′ adapter, at least one single-stranded nucleic acid blocker having a 5′ end of the blocker and a 3′ end of the blocker, and at least one second ligase to form a second reaction composition and incubating under conditions suitable for second ligation products to be formed, wherein the at least one single-stranded nucleic acid blocker is added before, simultaneously with, or after the 5′ adapter, and wherein the second reaction composition is incubated under conditions suitable for the single-stranded nucleic acid blocker to anneal with the undesired RNA or first ligation products comprising undesired RNA to form an RNA-blocker duplex, wherein the RNA-blocker duplex has a blunt duplex end comprising the 5′ end of the undesired RNA and the 3′ end of the blocker, or wherein the blocker extends, in single-stranded form, beyond the 5′ end of the undesired RNA in the RNA-blocker duplex, wherein the RNA-blocker duplex is formed before or simultaneously with the forming of the second ligation products;
• and
• amplifying the second ligation products to generate a pool of amplification products, wherein amplification products corresponding to the at least one undesired RNA species are depleted.

A method for depleting at least one undesired RNA species having a 5′ end of the undesired RNA and a 3′ end of the undesired RNA, comprising:

• combining a sample with at least one 3′ adapter and at least one first ligase to form a first reaction composition and incubating under conditions suitable for first ligation products to be formed;
• separating the first ligation products from the first reaction composition;
• combining the separated first ligation products with at least one 5′ adapter, at least one single-stranded nucleic acid blocker having a 5′ end of the blocker and a 3′ end of the blocker, and at least one second ligase to form a second reaction composition and incubating under conditions suitable for second ligation products to be formed, wherein the at least one single-stranded nucleic acid blocker is added before, simultaneously with, or after the 5′ adapter, and wherein the second reaction composition is incubated under conditions suitable for the single-stranded nucleic acid blocker to anneal with the undesired RNA or first ligation products comprising undesired RNA to form an RNA-blocker duplex, wherein the RNA-blocker duplex has a blunt duplex end comprising the 5″ end of the undesired RNA and the 3′ end of the blocker, or wherein the blocker extends, in single-stranded form, beyond the 5′ end of the undesired RNA in the RNA-blocker duplex, wherein the RNA-blocker duplex is formed before or simultaneously with the forming of the second ligation products;
• and
• amplifying the second ligation products to generate a pool of amplification products, wherein amplification products corresponding to the at least one undesired RNA species are depleted.