Droplet‐based single cell sequencing technologies, such as inDrop, Drop‐seq, and 10X Genomics, are catalyzing a revolution in the understanding of biology. Barcoding beads are key components for these technologies. What is limiting today are barcoding beads that are easy to fabricate, can efficiently deliver primers into drops, and thus achieve high detection efficiency. Here, this work reports an approach to fabricate dissolvable polyacrylamide beads, by crosslinking acrylamide with disulfide bridges that can be cleaved with dithiothreitol. The beads can be rapidly dissolved in drops and release DNA barcode primers. The dissolvable beads are easy to synthesize, and the primer cost for the beads is significantly lower than that for the previous barcoding beads. Furthermore, the dissolvable beads can be loaded into drops with >95% loading efficiency of a single bead per drop and the dissolution of beads does not influence reverse transcription or the polymerase chain reaction (PCR) in drops. Based on this approach, the dissolvable beads are used for single cell RNA and protein analysis.more » « less
- Award ID(s):
- NSF-PAR ID:
- Publisher / Repository:
- Wiley Blackwell (John Wiley & Sons)
- Date Published:
- Journal Name:
- Advanced Science
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
null (Ed.)Here, we demonstrate use of a Mg 2+ -dependent, site-specific DNA enzyme (DNAzyme) to cleave oligos from polyacrylamide gel beads, which is suitable for use in drop-based assays. We show that cleavage efficiency is improved by use of a tandem-repeat cleavage site. We further demonstrate that DNAzyme-released oligos function as primers in reverse transcription of cell-released mRNA.more » « less
Kolawole, Abimbola O. (Ed.)ABSTRACT Drop-based microfluidics has revolutionized single-cell studies and can be applied toward analyzing tens of thousands to millions of single cells and their products contained within picoliter-sized drops. Drop-based microfluidics can shed insight into single-cell virology, enabling higher-resolution analysis of cellular and viral heterogeneity during viral infection. In this work, individual A549, MDCK, and siat7e cells were infected with influenza A virus (IAV) and encapsulated into 100-μm-size drops. Initial studies of uninfected cells encapsulated in drops demonstrated high cell viability and drop stability. Cell viability of uninfected cells in the drops remained above 75%, and the average drop radii changed by less than 3% following cell encapsulation and incubation over 24 h. Infection parameters were analyzed over 24 h from individually infected cells in drops. The number of IAV viral genomes and infectious viruses released from A549 and MDCK cells in drops was not significantly different from bulk infection as measured by reverse transcriptase quantitative PCR (RT-qPCR) and plaque assay. The application of drop-based microfluidics in this work expands the capacity to propagate IAV viruses and perform high-throughput analyses of individually infected cells. IMPORTANCE Drop-based microfluidics is a cutting-edge tool in single-cell research. Here, we used drop-based microfluidics to encapsulate thousands of individual cells infected with influenza A virus within picoliter-sized drops. Drop stability, cell loading, and cell viability were quantified from three different cell lines that support influenza A virus propagation. Similar levels of viral progeny as determined by RT-qPCR and plaque assay were observed from encapsulated cells in drops compared to bulk culture. This approach enables the ability to propagate influenza A virus from encapsulated cells, allowing for future high-throughput analysis of single host cell interactions in isolated microenvironments over the course of the viral life cycle.more » « less
ABSTRACT In today’s technology, organ transplantation is found very challenging as it is not easy to find the right donor organ in a short period of time. In the last several decades, tissue engineering was rapidly developed to be used as an alternative approach to the organ transplantation. Tissue engineering aims to regenerate the tissues and also organs to help patients who waits for the organ transplantation. Recent research showed that in order to regenerate the tissues, cells must be seeded onto the 3D artificial laboratory fabricated matrices called scaffolds. If cells show healthy growth within the scaffolds, they can be implanted to the injured tissue to do the regeneration. One of the biggest limitation that reduces the success rate of tissue regeneration is the fabrication of accurate thick 3D scaffolds. In this research “maskless photolithography” was used to fabricate the scaffolds. Experiment setup consist of digital micro-mirror devices (DMD) (Texas Instruments, DLi4120), optical lens sets, UV light source (DYMAX, BlueWave 200) and PEGDA which is a liquid form photo-curable solution. In this method, scaffolds are fabricated in layer-by-layer fashion to control the interior architecture of the scaffolds. Working principles of the maskless photolithography is, first layer shape is designed with AutoCAD and the designed image is uploaded to the DMD as a bitmap file. DMD consists of hundreds of tiny micro-mirrors. When the UV light is turned on and irradiated the DMD, depending on the micro-mirrors’ angles, UV light is selectively reflected to the low percentage Polyethylene (glycol) Diacrylate (PEGDA) photo-curable solution. When UV light penetrates into the PEGDA, only the illuminated part is solidified and non-illuminated area still remains in the liquid phase. In this research, scaffolds were fabricated in three layers. First layer and the last layer are solid layers and y-shape open structure was sandwiched between these layers. After the first layer is fabricated with DMD, a “y-shape” structure was fabricated with the 3D printer by using the dissolvable filament. Then, it was placed onto the first solid layer and covered with fresh high percentage PEGDA solution. UV light was reflected to the PEGDA solution and solidified to make the second and third layers. After the scaffold was fabricated, it is dipped into the limonene solution to dissolve the y-shape away. Our results show that thick scaffolds can be fabricated in layer-by-layer fashion with “maskless photolithography”. Since the UV light is stable and does not move onto the PEGDA, entire scaffold can be fabricated in one single UV shot which makes the process faster than the current fabrication techniques.more » « less
Molecular ecologists frequently use genome reduction strategies that rely upon restriction enzyme digestion of genomic DNA to sample consistent portions of the genome from many individuals (e.g., RADseq, GBS). However, researchers often find the existing methods expensive to initiate and/or difficult to implement consistently, especially because it is difficult to multiplex sufficient numbers of samples to fill entire sequencing lanes. Here, we introduce a low-cost and highly robust approach for the construction of dual-digest RADseq libraries that build on adapters and primers designed in
Adapterama I. Major features of our method include: (1) minimizing the number of processing steps; (2) focusing on a single strand of sample DNA for library construction, allowing the use of a non-phosphorylated adapter on one end; (3) ligating adapters in the presence of active restriction enzymes, thereby reducing chimeras; (4) including an optional third restriction enzyme to cut apart adapter-dimers formed by the phosphorylated adapter, thus increasing the efficiency of adapter ligation to sample DNA, which is particularly effective when only low quantity/quality DNA samples are available; (5) interchangeable adapter designs; (6) incorporating variable-length internal indexes within the adapters to increase the scope of sample indexing, facilitate pooling, and increase sequence diversity; (7) maintaining compatibility with universal dual-indexed primers and thus, Illumina sequencing reagents and libraries; and, (8) easy modification for the identification of PCR duplicates. We present eight adapter designs that work with 72 restriction enzyme combinations. We demonstrate the efficiency of our approach by comparing it with existing methods, and we validate its utility through the discovery of many variable loci in a variety of non-model organisms. Our 2RAD/3RAD method is easy to perform, has low startup costs, has increased utility with low-concentration input DNA, and produces libraries that can be highly-multiplexed and pooled with other Illumina libraries.
Real‐time tracking of multiple particles is key for quantitative analysis of dynamic biophysical processes and materials science via time‐lapse microscopy image data, especially for single molecule biophysical techniques, such as magnetic tweezers and centrifugal force microscopy. However, real‐time multiple particle tracking with high resolution is limited by the current imaging processes or tracking algorithms. Here, we demonstrate 1 nm resolution in three dimensions in real‐time with a graphics‐processing unit (GPU) based on a compute unified device architecture (CUDA) parallel computing framework instead of only a central processing unit (CPU). We also explore the trade‐offs between processing speed and size of the utilized regions of interest and a maximum speedup of 137 is achieved with the GPU compared with the CPU. Moreover, we utilize this method with our recently self‐built centrifugal force microscope (CFM) in experiments that track multiple DNA‐tethered particles. Our approach paves the way for high‐throughput single molecule techniques with high resolution and efficiency.
Particles are widely used as probes in life sciences through their motions. In single molecule techniques such as optical tweezers and magnetic tweezers, microbeads are used to study intermolecular or intramolecular interactions via beads tracking. Also tracking multiple beads’ motions could study cell–cell or cell–ECM interactions in traction force microscopy. Therefore, particle tracking is of key important during these researches. However, parallel 3D multiple particle tracking in real‐time with high resolution is a challenge either due to the algorithm or the program. Here, we combine the performance of CPU and CUDA‐based GPU to make a hybrid implementation for particle tracking. In this way, a speedup of 137 is obtained compared the program before only with CPU without loss of accuracy. Moreover, we improve and build a new centrifugal force microscope for multiple single molecule force spectroscopy research in parallel. Then we employed our program into centrifugal force microscope for DNA stretching study. Our results not only demonstrate the application of this program in single molecule techniques, also indicate the capability of multiple single molecule study with centrifugal force microscopy.