Nanobody-tethered transposition enables multifactorial chromatin profiling at single-cell resolution
For NTT-seq with surface markers readout on primary cells, 1 million thawed PBMCs were resuspended in 200 µl of staining buffer and incubated for 15 minutes with 20 µl of Fc receptor block on ice. Cells were then washed three times with 1 ml of staining buffer and pooled together. The panel of oligo-conjguated antibodies was added to the cells to incubate for 30 minutes on ice.
For NTT-seq on PBMCs and BMMCs, 16% methanol-free formaldehyde was added for fixation at room temperature for 5 minutes. The cross-linking reaction was stopped by the addition of 12 µl of 1.25 M glycine solution. Subsequently, cells were washed twice with PBS. The permeabilization was performed by adding isotonic lysis buffer on ice for 7 minutes.
ADTs: during silane bead elution , beads were eluted in 43.5 μl of elution solution I. The extra 3 μl was used for the surface protein tags library. During SPRI cleanup , the supernatant was saved, and the short DNA derived from antibody oligos was purified with 2× SPRI beads. The eluted DNA was combined with the 3 µl left aside after the silane purification to be used as input for protein tag amplification.
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