Journal: Advanced Science

Article Title: Microbiome Single Cell Atlases Generated with a Commercial Instrument

doi: 10.1002/advs.202409338

Figure Lengend Snippet: EASi‐seq workflow: Genome purification, microfluidics, and bioinformatics. A) Microbial cells are suspended in a hydrogel precursor solution (acrylamide monomer and N,N’‐bis (acryloyl) cystamine‐BAC crosslinker) and emulsified with fluorinated oil by passing the mixture through a syringe needle. Gelation embeds individual cells within hydrogel beads. B) Hydrogel beads are size‐selected via differential centrifugation. Beads are suspended in a density‐matching buffer (40% sucrose in PBS with 0.1% Tween 20) and centrifuged at 1000 × g for 10 min to pellet oversized beads. The supernatant is centrifuged at 3000 × g for 10 min to pellet the desired bead size (5–30 µm), which is collected as size‐selected hydrogel beads. C) Cells within hydrogel beads are lysed using a two‐step enzyme digestion. A cocktail of four enzymes digests cell walls, followed by Proteinase K treatment to digest proteins. The small pore size of the hydrogel allows diffusion of proteins and smaller molecules while immobilizing long DNA molecules. Washing removes cellular debris, leaving purified genomic DNA trapped within the beads. D) Genomic DNA within hydrogel beads is tagmented in droplets during the first Tapestri microfluidic module (bottom), followed by barcoding PCR in the second module (top), using barcode beads paired with each droplet. E) Sequencing the barcoded amplicons produces single‐cell shotgun reads for thousands of microbial cells. F) EASi‐seq supports high‐throughput microbiome analysis, including genome atlas construction, cluster‐based genome assembly, strain identification, and pathway analysis.

Article Snippet: 200 μL Mission Bio V2 barcoding beads washed with Tris buffer (pH 8.0) and resuspended in 10 m m Tris buffer containing 3.75% tween 20, 2.5 m m MgCl 2 , 0.625 mg mL −1 BSA.

Techniques: Purification, Centrifugation, Pore Size, Diffusion-based Assay, Sequencing, High Throughput Screening Assay