Supporting data: 3D projection electrophoresis for single-cell immunoblotting (Part 2)

Immunoassays and mass spectrometry are powerful single-cell protein analysis tools; however, interfacing and throughput bottlenecks remain. Here, we introduce three-dimensional single-cell immunoblots to detect both cytosolic and nuclear proteins. The 3D microfluidic device is a photoactive polyacrylamide gel with a microwell array-patterned face (x-y) for cell isolation and lysis. Single-cell lysate in each microwell is ‘electrophoretically projected’ into the 3rd dimension (z-axis), separated by size, and photo-captured in the gel for immunoprobing and confocal/light-sheet imaging. Design and analysis are informed by the physics of 3D diffusion. Electrophoresis throughput is >2.5 cells/s (70X faster than published serial sampling), with 25 immunoblots/mm2 device area (>10X increase over previous immunoblots). The 3D microdevice design synchronizes analyses of hundreds of cells, compared to status quo serial analyses that impart hours-long delay between the first and last cell. Here, we introduce projection electrophoresis to augment the heavily genomic and transcriptomic single-cell atlases with protein-level profiling.

Methods
Acquisition: This dataset was acquired using a Zeiss Lightsheet Z.1 system fitted with a 5X detection objective (Zeiss EC Plan-Neofluar 420330-8210) and 4X light sheet forming objectives (Zeiss LSFM 400900-9010).  During imaging, the polyacrylamide gel sample was mounted to a coverslip and immersed in 1X Tris-buffered Saline with Tween (details provided in the Methods section of the manuscript).

Analysis: Analysis scripts for projection electrophoresis data are available in the following GitHub repository: https://github.com/samanthagrist/projection_ep_analysis.  Acquisition and processing details for each portion of the dataset are available in the relevant README files included in the relevant subdirectories of the main portion of the dataset (Supporting data: 3D projection electrophoresis for single-cell immunoblotting).