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Dissolvable Gel Beads Containing Oligo DNA

Preparation of dissolvable gel beads containing Oligo DNA with Microdroplet/Microsphere Generator

Experimental Purpose:In this application note, based on the barcode gel bead preparation method of 10X Genomics, the Microdroplet/Microsphere Generator is used to prepare highly monodisperse dissolvable polyacrylamide gel beads containing Oligo DNA (CV<5%). An acrylamide mixture solution (acrylamide, N, N’- bis (acryloyl) cystamine and primer solution) is used as the dispersed phase, and Drop-Surf Generation Oil (containing N, N, N’, N’- tetramethylethylenediamine) is used as the continuous phase. The cross-linking rate of Oligo DNA concentration is up to 40%.



As the basic unit of biological structure and function, cells differ greatly in morphological types. Combined with high-throughput sequencing, droplet-based single cell sequencing technologies have been successfully applied to analyzing RNA [1-3], DNA [4,5] and protein [6,7] in single cells. To track a single cell, it is necessary to encapsulate the gel beads containing DNA barcode together with the single cell in nanoliter-sized microdroplets for subsequent single cell analysis and sequencing [8]. The key to realize this technique is to add the synthesized DNA barcode to the gel beads.

Currently, there are several reported methods for preparing DNA barcode, including polyacrylamide gel beads used in inDrop[2], hydroxylated methacrylic acid polymer gel beads used in Drop-seq[3] and polyacrylamide gel beads used in 10X Genomics[8]. However, the gel beads produced by these methods has their own advantages and shortcomings. In the inDrop system, the gel beads can be closely packed in a microfluidic device channel to achieve more than 95% loading of single bead per drop. However, the release of DNA barcode in the process requires ultraviolet rays, which increases the difficulty of primer release[2]. Also, ultraviolet rays may also cause irreversible damage to DNA or RNA. In the Drop-seq system, the primers cannot be released from the beads, which means the reaction only take place on the surface of the beads and reduces the reaction efficiency. In 10X Genomics system, the beads can be effectively encapsulated and released, but the relatively high cost and the lack of flexibility in primer design on beads limit the development of some experiments. What thus missing today, is barcode beads that are easy to fabricate and efficiently deliver primers into drops, so as to achieve high detection efficiency.

Based on Wang et al.’s research [9] and the preparation method of barcode gel beads of 10X Genomics, FluidicLab provides an effective strategy to produce dissolvable polyacrylamide gel beads containing Oligo DNA. The schematic diagram is shown in the follow picture. The dispersed phase (containing acrylamide, ammonium persulfate, N, N’- bis (acryloyl) cystamine and primer solution), and the continuous phase (Drop-Surf Droplet Generation Oil and N, N, N’, N’- tetramethylethylenediamine) are pushed into a microfluidic chip by FluidicLab Microdroplet Generator, and droplets are generated and collected to obtain highly monodisperse dissolvable polyacrylamide gel beads containing Oligo DNA. The gel beads can be completely dissolvable by opening its disulfide bond with dithiol, resulting in releasing DNA barcode.

Preparation of dissolvable gel beads containing Oligo DNA with Microdroplet/Microsphere Generator



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