Thermomixer c 15

Roughly 3,000 DART-seq beads were added to a mixture containing 18 uL of 5M NaCl, 2 μL of 1M Tris HCl pH 8.0, 1 μL of SDS, 78 μl of water, and 1 μL of 100 μM Cy5 fluorescently labeled oligo (see Supplementary Table). The beads were incubated for 45 minutes at 46 ˚C in an Eppendorf ThermoMixer C (15”, at 1800 RPM). Following incubation, the beads were pooled and washed with 250 μL TE-SDS, followed by 250 μL TE-TW. The beads were suspended in 200 μl of DNAse/RNAse free water and transferred to a Qubit assay tube (ThermoFisher Scientific, Q32856). Qubit 3.0 Fluorometer was set to “Fluorometer” mode under the “635 nm” emission setting. The tube was vortexed briefly and placed in the fluorometer for immediate readout. Two additional vortexing and measurement steps were performed.

Roughly 6,000 DART-seq beads were added to a mixture containing 18 uL of 5M NaCl, 2 μL of 1M Tris HCl pH 8.0, 1 μL of SDS, 78 μl of water, and 1 μL of 100 μM Cy5 fluorescently labeled oligo (see Supplementary Table). The beads were incubated for 45 minutes at 46 ˚C in an Eppendorf ThermoMixer C (15”, at 1800 RPM). Following incubation, the beads were pooled and washed with 250 μL TE-SDS, followed by 250 μL TE-TW. The beads were suspended in water and imaged in the Zeiss Axio Observer Z1 in the Cy5 channel and bright field. A custom Python script was used to determine the fluorescence intensity of each bead.