The present disclosure relates to processes for inverting oligonucleotide probes in an in situ synthesized array. These processes can be used to reverse the orientation of probes with respect to the substrate from 3-bound to a substrate to 5-bound to another substrate. These processes can also be used to reduce or eliminate the presence of truncated probe sequences from an in situ synthesized array. These processes can preserve the original patterns of the synthesized oligonucleotide after the inversion. These process can be achieved via the formation of a hydrogel layer in-between a donor substrate and an acceptor substrate through a polymerization reaction forming the hydrogel layer.

The present disclosure relates to processes for suspending stretched nucleic acids over surface features. These processes can be used to prepare stretched nucleic acids that are more active in enzymatic reactions and other reactions than those laid down on a flat surface. These processes can be achieved by using a photoresist layer on top of a substrate, stretch a nucleic acid on top of the surface, and then remove part of the photoresist to form surface features that suspend the stretched nucleic acid. Furthermore, the formation of a hydrogel layer over the stretched nucleic acid and the surface features can transfer the stretched nucleic acid to the hydrogel for further reactions, including enzymatic reactions.

The present disclosure relates to processes for suspending stretched nucleic acids over surface features. These processes can be used to prepare stretched nucleic acids that are more active in enzymatic reactions and other reactions than those laid down on a flat surface. These processes can be achieved by using a photoresist layer on top of a substrate, stretch a nucleic acid on top of the surface, and then remove part of the photoresist to form surface features that suspend the stretched nucleic acid. Furthermore, the formation of a hydrogel layer over the stretched nucleic acid and the surface features can transfer the stretched nucleic acid to the hydrogel for further reactions, including enzymatic reactions.

The invention is directed to methods for synthesizing oligonucleotides direction on biomolecules or cells living or fixed. In some embodiments, template-free enzymatic synthesis is implemented under biological conditions with successive cycles of (i) enzymatic addition of a 3-O-blocked nucleoside triphosphate and (ii) enzymatic deblocking of the incorporated nucleotide to regenerate a free 3 hydroxyl. The invention has applications in single-cell cDNA library construction and analysis.

Embodiments in accordance with the present disclosure are directed to polymer beads and uses thereof, including forming libraries of compounds for screening and assay purposes. A polymer bead, in accordance with embodiments, has an interior surface and an exterior surface that are topologically segregated from one another. The interior surface includes a protecting group and the exterior surface includes a deprotected group, which can also be referred to as a deprotected functional group. The protecting group can includes a nitrobenzenesulfonamide group that protects an amine group.

Disclosed herein are formulations, substrates, and arrays. In certain embodiments, substrates and arrays comprise a porous layer for synthesis and attachment of polymers or biomolecules. Also disclosed herein are methods for manufacturing and using the formulations, substrates, and arrays, including porous arrays. Also disclosed herein are formulations and methods for one-step coupling, e.g., for synthesis of peptides in an N->C orientation. In some embodiments, disclosed herein are formulations and methods for high efficiency coupling of biomolecules to a substrate.

Disclosed herein are formulations, substrates, and arrays. In certain embodiments, substrates and arrays comprise a porous layer for synthesis and attachment of polymers or biomolecules. Also disclosed herein are methods for manufacturing and using the formulations, substrates, and arrays, including porous arrays. Also disclosed herein are formulations and methods for one-step coupling, e.g., for synthesis of peptides in an N->C orientation. In some embodiments, disclosed herein are formulations and methods for high efficiency coupling of biomolecules to a substrate.

The present disclosure provides methods of generating supports (e.g., beads) comprising barcode molecules coupled thereto. A barcode molecule coupled to a support may comprise a barcode sequence and a functional sequence. A barcode molecule may be generated using two or more ligation reactions in a combinatorial fashion. A support comprising two or more different barcode molecules may be useful for analyzing or processing one or more analytes such as nucleic acid molecules, proteins, and/or perturbation agents.

The present disclosure provides methods of generating supports (e.g., beads) comprising barcode molecules coupled thereto. A barcode molecule coupled to a support may comprise a barcode sequence and a functional sequence. A barcode molecule may be generated using two or more ligation reactions in a combinatorial fashion. A support comprising two or more different barcode molecules may be useful for analyzing or processing one or more analytes such as nucleic acid molecules, proteins, and/or perturbation agents.

The present disclosure provides methods and processes for forming a pattern of oligonucleotides on a microarray. A method for forming a pattern of oligonucleotides on a microarray may include forming a photoresist layer by applying a photoresist composition onto an underlying layer of a substrate, exposing a dose of light through a patterned mask onto the substrate, and removing protective groups on a section of the plurality of functional groups within at least one exposed region of the substrate, wherein the photoresist composition comprises a photoacid generator, an acid scavenger and a photosensitizer, wherein the underlying layer comprises a plurality of functional groups protected by protective groups; thereby forming a pattern on the substrate, wherein the pattern comprises the at least one exposed region, and wherein the at least one exposed region is no more than 1 micrometer in at least one dimension.