DOI: 10.6078/d1n989
title: Supporting data: 3D projection electrophoresis for single-cell immunoblotting (Part 2)

description: 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 &gt;2.5 cells/s
 (70X faster than published serial sampling), with 25
 immunoblots/mm2 device area (&gt;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.
description: <strong>Acquisition</strong>: 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).
 <strong>Analysis</strong>:
 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).
description: <strong>Data
 Overview</strong><br> This dataset contains the
 tiled light sheet microscopy image (Zeiss .czi format) of  the 'Gel
 6' separation (separated 2019-09-10) probed and imaged 2019-10-03
 ('Gel 1' in Figure 5).  For this dataset, the sample was imaged
 in the fluorescence channels corresponding to the probed GAPDH (AlexaFluor
 488) and Actinin (AlexaFluor 555) proteins (relevant to Figure 5 and
 Supplementary Figures 4-6). The large .czi file was
 split into pieces for upload using Rclone (https://rclone.org/chunker/). 
 All of the files with the same base filename can be sequentially
 concatenated (e.g. with 'cat' or Rclone) to reconstitute the
 original large file.  An example workflow on MacOS is as
 follows: Download all of the chunked files
 into a directory. In Terminal, navigate to the
 directory containing the chunked files. Run the
 following command to concatenate the files: <br>
 cat 2019-09-10_Gel6_EPProbed_Actinin555GAPDH488.czi.rclone_chunk.0*
 &gt; 2019-09-10_Gel6_EPProbed_Actinin555GAPDH488_Merged.czi
 Open the merged file by dragging and dropping onto Fiji and
 selecting the Bio-Formats import parameters, or by opening in Zeiss Zen
 Lite. <strong>Linked
 Data</strong><br> Additional data are available in
 separate, linked data publications due to file size constraints:
 Analyzed data and additional README files
 corresponding to this dataset are presented in the main data publication
 for this manuscript: 'Supporting data: 3D projection electrophoresis
 for single-cell immunoblotting' (DOI: 10.6078/D1B13V).  That data
 publication's README files mention this dataset where
 relevant. Tiled light sheet microscopy image for the
 'Gel 5' separation probed and imaged 2019-10-03 ('Gel
 2' in Figure 5), imaged in the GAPDH and Actinin protein channels
 (DOI: 10.6078/D1811G). Tiled light sheet microscopy
 image for the 'Gel 5' separation probed and imaged 2019-10-03
 ('Gel 2' in Figure 5), imaged in the GAPDH and Beta Tubulin
 protein channels (DOI: 10.6078/D1HH6C, relevant only to Supplementary
 Figure 6). Please see the Related Works
 for links to these additional datasets.
 <strong>Analysis</strong><br> Analysis scripts to analyze light sheet microscopy images of projection electrophoresis separations are available in the following GitHub repository: https://github.com/samanthagrist/projection_ep_analysis (in the 'single-cell-lightsheet-analysis' subdirectory).  Initialization files and analyzed data files for the analysis scripts corresponding to this dataset are included in the main data publication for this manuscript (in the 'SI_Fig6_Single_Cell_Migration_Comparison/lightsheet/ subdirectory)