Single-cell RNA sequencing (scRNA-seq) technologies are poised to reshape the current cell-type classification system. However, a transcriptome-based single-cell atlas has not been achieved for complex mammalian systems. Here, we developed Microwell-seq, a high-throughput and low-cost scRNA-seq platform using simple, inexpensive devices. Using Microwell-seq, we analyzed more than 400,000 single cells covering all of the major mouse organs and constructed a basic scheme for a mouse cell atlas (MCA). We reveal a single-cell hierarchy for many tissues that have not been well characterized previously. We built a web-based "single-cell MCA analysis" pipeline that accurately defines cell types based on single-cell digital expression. Our study demonstrates the wide applicability of the Microwell-seq technology and MCA resource.
It has come to our attention that in preparing the final version of this paper, we inadvertently misspelled the first name of an author Ziming Zhou as ''Zimin Zhou''. In addition, we have made two errors in describing the reagents in the STAR Methods. First, under the subheading of ''Synthesis of barcoded beads'' in the Method Details section, the supplier of the magnetic beads coated with carboxyl groups should be Suzhou Knowledge & Benefit Sphere Tech. Co., Ltd. (diameter 20-25 mm, http://www.kbspheretech. com/), instead of Zhiyi. Second, under the subheading of ''Cell collection and lysis'' in the Method Details section, the concentration of Tris-HCL for the cold lysis buffer should be 0.1 M, instead of 1 M. These errors have been corrected online, and we apologize for any confusions we may have caused.
Single cell proteomic analysis provides crucial information on cellular heterogeneity in biological systems. Herein, we describe a nanoliter-scale oil-air-droplet (OAD) chip for achieving multistep complex sample pretreatment and injection for single cell proteomic analysis in the shotgun mode. By using miniaturized stationary droplet microreaction and manipulation techniques, our system allows all sample pretreatment and injection procedures to be performed in a nanoliter-scale droplet with minimum sample loss and a high sample injection efficiency (>99%), thus substantially increasing the analytical sensitivity for single cell samples. We applied the present system in the proteomic analysis of 100 ± 10, 50 ± 5, 10, and 1 HeLa cell(s), and protein IDs of 1360, 612, 192, and 51 were identified, respectively. The OAD chip-based system was further applied in single mouse oocyte analysis, with 355 protein IDs identified at the single oocyte level, which demonstrated its special advantages of high enrichment of sequence coverage, hydrophobic proteins, and enzymatic digestion efficiency over the traditional in-tube system.
A simple, room-temperature bonding process was developed for the fabrication of glass microfluidic chips. High-quality bonding with high yields (>95%) was achieved without the requirement of clean room facilities, programmed high-temperature furnaces, pressurized water sources, adhesives, or pressurizing weights. The plates to be bonded were sequentially prewashed with acetone, detergent, high-flow-rate (10-20 m/s) tap water, and absolute ethyl alcohol and were soaked in concentrated sulfuric acid for 8-12 h. The plates were again washed in high-flow-rate tap water for 5 min and, finally, with demineralized water. The plates were bonded by bringing the cleaned surfaces into close contact under a continuous flow of demineralized water and air-dried at room temperature for more than 3 h. This bonding process features simple operation, good smoothness of the plate surface, and high bonding yield. The procedures can be readily applied in any routine laboratory. The bonding strength of glass chips thus produced, measured using a shear force testing procedure, was higher than 6 kg/cm(2). The mechanism for the strong bonding strength is presumably related to the formation of a hydrolyzed layer on the plate surfaces after soaking the substrates in acid or water for extended periods. Microfluidic chips bonded by the above procedure were tested in the CE separation of fluorescein isothiocyanate-labeled amino acids.
This contribution describes a sequential operation droplet array (SODA) system, a fully automated droplet-based microfluidic system capable of performing picoliter-scale liquid manipulation, analysis, and screening. The SODA system was built using a tapered capillary-syringe pump module and a two-dimensional (2D) oil-covered droplet array installed on an x-y-z translation stage. With the system, we developed a novel picoliter-scale droplet depositing technique for forming a 2D picoliter-droplet array. On this basis, an automated droplet manipulation method with picoliter precision was established using the programmable combination of the capillary-based liquid aspirating-depositing and the moving of the oil-covered droplet array, the so-called "aspirating-depositing-moving" (ADM) method. Differing from the previously reported droplet systems based on microchips, microcapillaries, or digital microfluidics, this method can achieve complete and flexible droplet manipulations, including droplet assembling, generation, indexing, transferring, splitting, and fusion in the picoliter range, endowing the present system with ultralow sample/reagent consumptions and substantial versatility in analysis and screening for multiple different samples. To demonstrate its feasibility and versatility, we applied the SODA system in multiple experiments required in drug screening, including the screening of inhibitors for capases-1 from a chemical library, the measurement of IC50 values for the identified inhibitors, and the screening of the synergistic effect of multiple inhibitors. In the experiments, the consumptions of samples and reagents are only 60-180 pL for each droplet microreactor, which are commonly 3-5 orders of magnitude lower than those of conventional multiwell plate systems, and 1-2 orders of magnitude lower than other droplet-based microfluidic systems for multiple sample screening. The ability of the SODA system in performing complicated and multistep droplet manipulations was further demonstrated in the serial dilution of nanoliter-scale inhibitor droplets with concentrations spanning 6 orders of magnitude for IC50 profiling, which includes droplet generation, indexing, splitting, transferring, and fusion with picoliter precision.
We performed cell-based drug combination screening using an integrated droplet-based microfluidic system based on the sequential operation droplet array (SODA) technique. In the system, a tapered capillary connected with a syringe pump was used for multistep droplet manipulations. An oil-covered two-dimensional droplet array chip fixed in an x-y-z translation stage was used as the platform for cell culture and analysis. Complex multistep operations for drug combination screening involving long-term cell culture, medium changing, schedule-dependent drug dosage and stimulation, and cell viability testing were achieved in parallel in the semiopen droplet array, using multiple droplet manipulations including liquid metering, aspirating, depositing, mixing, and transferring. Long-term cell culture as long as 11 days was performed in oil-covered 500 nL droplets by changing the culture medium in each droplet every 24 h. The present system was applied in parallel schedule-dependent drug combination screening for A549 nonsmall lung cancer cells with the cell cycle-dependent drug flavopiridol and two anticancer drugs of paclitaxel and 5-fluorouracil. The highest inhibition efficiency was obtained with a schedule combination of 200 nM flavopiridol followed by 100 μM 5-fluorouracil. The drug consumption for each screening test was substantially decreased to 5 ng-5 μg, corresponding to 10-1000-fold reductions compared with traditional drug screening systems with 96-well or 384-well plates. The present work provides a novel and flexible droplet-based microfluidic approach for performing cell-based screening with complex and multistep operation procedures.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.