RESUMO
We introduce a simple integrated analysis method that links cellular phenotypic behaviour with single-cell RNA sequencing (scRNA-seq) by utilizing a combination of optical indices from cells and hydrogel beads. With our method, the combinations, referred to as joint colour codes, enable the link via matching the optical combinations measured by conventional epi-fluorescence microscopy with the concatenated DNA molecular barcodes created by cell-hydrogel bead pairs and sequenced by next-generation sequencing. We validated our approach by demonstrating an accurate link between the cell image and scRNA-seq with mixed species experiments, longitudinal cell tagging by electroporation and lipofection, and gene expression analysis. Furthermore, we extended our approach to multiplexed chemical transcriptomics, which enabled us to identify distinct phenotypic behaviours in HeLa cells treated with various concentrations of paclitaxel, and determine the corresponding gene regulation associated with the formation of a multipolar spindle.
Assuntos
Perfilação da Expressão Gênica , Transcriptoma , Humanos , Células HeLa , Perfilação da Expressão Gênica/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Hidrogéis , Análise de Célula Única/métodos , Análise de Sequência de RNA/métodosRESUMO
The development of artificial spider silk with properties similar to native silk has been a challenging task in materials science. In this study, we use a microfluidic device to create continuous fibers based on recombinant MaSp2 spidroin. The strategy incorporates ion-induced liquid-liquid phase separation, pH-driven fibrillation, and shear-dependent induction of ß-sheet formation. We find that a threshold shear stress of approximately 72 Pa is required for fiber formation, and that ß-sheet formation is dependent on the presence of polyalanine blocks in the repetitive sequence. The MaSp2 fiber formed has a ß-sheet content (29.2%) comparable to that of native dragline with a shear stress requirement of 111 Pa. Interestingly, the polyalanine blocks have limited influence on the occurrence of liquid-liquid phase separation and hierarchical structure. These results offer insights into the shear-induced crystallization and sequence-structure relationship of spider silk and have significant implications for the rational design of artificially spun fibers.
Assuntos
Fibroínas , Aranhas , Animais , Seda/química , Microfluídica , Fibroínas/química , Sequências Repetitivas de Ácido NucleicoRESUMO
The extraction of cellular contents from plant cells covered with cell walls remains a challenge, as it is physically hindered by the cell wall. We present a new microfluidic approach that leverages an intense pulsed electric field for permeabilizing the cell wall and a focused DC electric field for extracting the cellular contents selectively from a few targeted cells in a cluster of intact plant cells. We coupled the approach with on-chip fluorescence quantification of extracted molecules leveraging isotachophoresis as well as off-chip reverse transcription-quantitative polymerase chain reaction detecting extracted mRNA molecules. Our approach offers a workflow of about 5 min, isolating a cluster of intact plant cells, permeabilizing the cell wall, selectively extracting cytosolic molecules from a few targeted cells in the cluster, and outputting them to off-chip analyses without any enzymatic reactions. We anticipate that this approach will create a new opportunity to explore plant biology through less biased data realized by the rapid extraction of molecules from intact plant clusters.
Assuntos
Isotacoforese , Parede Celular , Microfluídica , Análise de Sequência com Séries de Oligonucleotídeos , PlantasRESUMO
We present a microfluidic method for electrical lysis and RNA extraction from single fixed cells leveraging reversible cross-linker dithiobis(succinimidyl propionate) (DSP). Our microfluidic system captures a single DSP-fixed cell at a hydrodynamic trap, reverse-cross-links the DSP molecules on a chip with dithiothreitol, lyses the plasma membrane via electrical field, and extracts cytoplasmic RNA with isotachophoresis-aided nucleic acids extraction. All of the on-chip processes complete in less than 5 min. We demonstrated the method using K562 leukemia cells and benchmarked the performance of RNA extraction with reverse transcription quantitative polymerase chain reaction. We also demonstrated the integration of our method with single-cell RNA sequencing.