v2 barcoding beads  (Mission Bio)

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

Journal: bioRxiv

Article Title: Microbiome single cell atlases generated with a commercial instrument

doi: 10.1101/2023.08.08.551713

Figure Lengend Snippet: a) Microbial cells suspended in hydrogel precursor (acrylamide monomer and BAC crosslinker) are emulsified with a fluorinated oil by passing the mixture through a syringe needle. After gelation, the cells are individually embedded in hydrogel beads. b) The hydrogel beads are size selected using differential centrifugation. The hydrogel beads are suspended in a density matching buffer (40% sucrose in PBS with 0.1% Tween 20) and centrifuged. Particles of different size sediment at different rates, with the larger particles sedimenting faster. After centrifuge at 1000 × g for 10 min, the oversized hydrogel beads are pelted, and the supernatants are subject to a centrifuge at a higher speed (3000xg for 10 min). The pellets are then collected as the size-selected hydrogel beads. c) Cells are lysed within the hydrogel beads by a two-step enzyme digestion. The beads are first subjected to a cocktail of 4 different enzymes that digest cell walls before being treated with protease K to digest proteins. The small pore-size of the hydrogel allow proteins and other molecules to freely diffuse, while immobilizing long DNA molecules. After the treatments and washing, only genomic DNA remains in the hydrogel beads. d) The microbial genomic DNA in each hydrogel bead is tagmented in a droplet (first step, bottom ) before being subsequently paired with barcode beads for barcoding PCR (second step, top ) on using the Tapestri instrument’s microfluidic modules. e) Sequencing of amplicons from the barcoding PCR generates single-cell shotgun reads for thousands of cells. f) EASi-seq allows high-throughput microbiome genome atlas analysis, as well as 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 mM Tris buffer containing 3.75% tween 20, 2.5 mM MgCl2, 0.625 mg/mL BSA.

Techniques: Centrifugation, Sequencing, High Throughput Screening Assay