title: Sequencing data for single cells and DNA-functionalized microbeads encapsulated in hydrogel droplets.
design: Sequencing data for single cells and DNA-functionalized microbeads encapsulated in hydrogel droplets.
library_construction_protocol: Micro-particles functionalized both by DNA barcodes and fluorescent molecules were synthesized and used for the study. Synthetic DNA oligonucleotides bearing barcodes sequences provided in protocol 13 were used in the study. DNA-barcoded microbeads were resuspended in a buffer containing 0.75% (w/v) agarose (Sigma Aldrich, ultra-low gelling temperature). Monodisperse droplets were generated by flowing the beads suspension and HFE7500 oil supplemented with 2% 008-fluorosurfactant (RAN Biotechnologies) into a droplet generation chip. After droplet generation, water-in-oil hydrogel droplets were cooled at 4 °C at 500 rpm shaking. To the droplets, medium-based buffer solution and perfluoro octanol (final volume: 20% in oil, v/v) were slowly added, and hydrogel droplets were extracted into the aqueous layer, which was further washed with 20% perfluorooctanol in HFE7200, and hexane supplemented with Span 80. The resulting hydrogel droplets were firstly subjected to microscopic imaging and then “single-droplet” sequencing, where the whole nucleic acid components in a hydrogel droplet were processed as single-cell components in the droplet-based single-cell RNA sequencing library preparation. Cell lysis and RNA extraction using polyT-functionalized DNA-barcoded beads for droplet-based single cell RNA sequencing (drop-seq) Reverse transcription and cDNA preparation using polyT-functionalized DNA-barcoded beads for droplet-based single cell RNA sequencing (drop-seq)