Oct 15, 2024

Public workspaceMultiplexed Surface Tethering of Extracellular Proteins (MultiSTEP) Protocols

DOI
dx.doi.org/10.17504/protocols.io.yxmvme8kog3p/v1
  • Nicholas A. Popp1,
  • Melinda K. Wheelock2,
  • Raining Wang2,
  • Rachel L. Powell2,
  • Douglas M. Fowler2
  • 1University of Washington Medical Scientist Training Program;
  • 2University of Washington
  • Atlas of Variant Effects Alliance
Sarah Heidl
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DOI: dx.doi.org/10.17504/protocols.io.yxmvme8kog3p/v1
Collection Citation: Nicholas A. Popp, Melinda K. Wheelock, Raining Wang, Rachel L. Powell, Douglas M. Fowler 2024. Multiplexed Surface Tethering of Extracellular Proteins (MultiSTEP) Protocols. protocols.io https://dx.doi.org/10.17504/protocols.io.yxmvme8kog3p/v1
License: This is an open access collection distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this collection and it's working
Created: October 04, 2024
Last Modified: October 15, 2024
Collection Integer ID: 109512
Keywords: Libaray cloning, Flow cytometry, Tissue culture, PCR amplification, Mutagenesis, Transfection
Funders Acknowledgement:
NHGRI (IGVF)
Grant ID: HG011969
Abstract
Multiplexed Surface Tethering of Extracellular Proteins (MultiSTEP) combines multiplexed human cell surface display with a genomically encoded landing pad to measure the effects of secreted protein variants at scale. The following protocols detail library cloning in the MultiSTEP system, tissue culture experiments, and measurement of variant abundance via flow cytometry.
Files
Protocol
Icon representing the file Site-saturation mutagenesis library cloning
Name
Site-saturation mutagenesis library cloning
Version 1
, University of Washington, Starita Lab
Sarah HeidlUniversity of Washington, Starita Lab
Protocol
Icon representing the file Barcoding site-saturation mutagenesis libraries
Name
Barcoding site-saturation mutagenesis libraries
Version 1
, University of Washington, Starita Lab
Sarah HeidlUniversity of Washington, Starita Lab
Protocol
Icon representing the file Illumina NGS sequencing of library plasmid
Name
Illumina NGS sequencing of library plasmid
Version 1
, University of Washington, Starita Lab
Sarah HeidlUniversity of Washington, Starita Lab
Protocol
Icon representing the file PacBio long-read sequencing
Name
PacBio long-read sequencing
Version 1
, University of Washington, Starita Lab
Sarah HeidlUniversity of Washington, Starita Lab
Protocol
Icon representing the file Purity sorting HEK293F cells
Name
Purity sorting HEK293F cells
Version 1
, University of Washington, Starita Lab
Sarah HeidlUniversity of Washington, Starita Lab
Protocol
Icon representing the file Library transfection
Name
Library transfection
Version 1
, University of Washington, Starita Lab
Sarah HeidlUniversity of Washington, Starita Lab
Protocol
Icon representing the file Staining Experiment and Sorting of 293F Cells
Name
Staining Experiment and Sorting of 293F Cells
Version 1
, University of Washington, Starita Lab
Sarah HeidlUniversity of Washington, Starita Lab
Protocol
Icon representing the file Barcode Sequencing by Illumina NGS
Name
Barcode Sequencing by Illumina NGS
Version 1
, University of Washington, Starita Lab
Sarah HeidlUniversity of Washington, Starita Lab
Protocol
Icon representing the file PCR amplification of the barcode region
Name
PCR amplification of the barcode region
Version 1
, University of Washington, Starita Lab
Sarah HeidlUniversity of Washington, Starita Lab