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: HEK293 cells were purchased from JCRB CellBank (JCRB9068); K562 cells were purchased from JCRB CellBank (JCRB0019). Murine NIH/3T3 cells (spiked only in sample 1) were purchased from JCRB CellBank (JCRB0615). K562 cells were grown in RPMI1640 medium (Sigma-Aldrich) supplemented with 10%FBS (Sigma-Aldrich) and 1x Antibiotic Antimycotic (Thermo Fisher Scientific). HEK293 cells were grown in Minimum Essential Medium Eagle (Sigma-Aldrich) supplemented with 10% Horse serum (Thermo Fisher Scientific) and 1x Antibiotic Antimycotic to reach 60-80% confluence. NIH/3T3 cells (used to estimate the rate of doublet errors during NGS library preparation, Fig. S5a) were grown in Dulbecco's Modified Eagles' Medium low glucose (Sigma-Aldrich) supplemented with 10% Newborn Calf serum (Thermo Fisher Scientific) and 1x Antibiotic Antimycotic to reach 60-80% confluence. Cell pellets were resuspended in a buffer containing  0.75% (w/v) agarose (Sigma Aldrich, ultra-low gelling temperature) and the suspension was passed through pluriStrainer (20 μm). 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)