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Authors: Marda Jorgensen
Group: Human BioMolecular Atlas Program (HuBMAP) Method Development Community
Summary: This SOP describes the methods used to ship paraffin-embedded blocks to the University of Zurich.
Proper citation: Marda Jorgensen 2020. Shipping Paraffin Blocks to the Bodenmiller Lab for IMC Analysis. protocols.io dx.doi.org/10.17504/protocols.io.bf5njq5e Copy
Authors: Sarah Urata, Blue Lake, Dinh Diep, Masato Hoshi, Sanjay Jain, Kun Zhang
Group: KPMP, Human BioMolecular Atlas Program (HuBMAP) Method Development Community
Summary: 10X Genomics Single Cell 3' (v3) RNA sequencing is a microdroplet-based method that permits the effective capture and sequencing of the mRNA and pre-mRNA molecules from single nuclei [1]. RNA molecules are transcribed and processed within the nucleus before exporting to ER for translation into proteins. As such, nuclear RNA is a mixture of nascent transcripts, partially or fully processed mRNA, and various non-coding RNA molecules. The total RNA content within the nucleus is roughly 10% of the RNA content in a whole cell, but has been found to accurately represent whole cell expression values in adult human tissues [2,3] including the kidney [4]. Nuclei can be readily isolated from frozen tissues with a combination of chemical and physical treatments that can effectively circumvent the non-uniform or incomplete dissociation of solid tissues into single cells, as well as RNA degradation or artefacts (such as stress response) during dissociation. Here we present a modified version of the published 10X protocol [1] that we have adapted for the processing of adult human kidney nuclei.References 1. Chromium Single Cell 3' Reagent Kits v3 User Guide (Rev A) CG000183, support.10xgenomics.com. 2. Lake et al. (2016). Science, doi:10.1126/science.aaf1204. 3. Lake et al. (2018). Nature Biotechnology, doi:10.1038/nbt.4038. 4. Lake et al. (2019). Nature Communications, doi:10.1038/s41467-019-10861-2.
Proper citation: Sarah Urata, Blue Lake, Dinh Diep, Masato Hoshi, Sanjay Jain, Kun Zhang 2019. 10X Genomics Single-Nucleus RNA-Sequencing for Transcriptomic Profiling of Adult Human Tissues. protocols.io dx.doi.org/10.17504/protocols.io.86khzcw Copy
Authors: John Hickey
Group: Human BioMolecular Atlas Program (HuBMAP) Method Development Community
Summary: See our detailed protocol published with the following title: CODEX multiplexed tissue imaging with DNA-conjugated antibodies.
Proper citation: John Hickey 2021. Reagent prep - Stanford TMC. protocols.io dx.doi.org/10.17504/protocols.io.be92jh8e Copy
Authors: Elizabeth Neumann, Jamie Allen, Maya Brewer, David Anderson, Mark De Caestecker, Danielle Gutierrez, Jeff Spraggins
Group: VU Biomolecular Multimodal Imaging Center, Human BioMolecular Atlas Program (HuBMAP) Method Development Community
Summary: We aim to develop high resolution, chemically informative imaginig methodologies for building an atlas of human organs, such as the kidney.Scope:Provide an overview of the methods used by the Vanderbilt Tissue Mapping Center as part of the Human Biomolecular Atlas Program (HuBMAP, NIH Common Fund) and contextualize individual protocols within our larger workflow.
Proper citation: Elizabeth Neumann, Jamie Allen, Maya Brewer, David Anderson, Mark De Caestecker, Danielle Gutierrez, Jeff Spraggins 2020. VU Biomolecular Multimodal Imaging Center (BIOMIC) kidney characterization pipeline for tissues collected through the Cooperative Human Tissue Network (CHTN). protocols.io dx.doi.org/10.17504/protocols.io.bb2wiqfe Copy
Authors: Danielle Gutierrez, Jamie Allen, Zach Jenkins, Jeff Spraggins
Group: VU Biomolecular Multimodal Imaging Center, Human BioMolecular Atlas Program (HuBMAP) Method Development Community
Summary: List of parameters and settings for searching label free proteomic data in MaxQuant version 1.6.7.
Proper citation: Danielle Gutierrez, Jamie Allen, Zach Jenkins, Jeff Spraggins 2020. LC-MS/MS Label-Free Proteomic Data Analysis Parameters. protocols.io dx.doi.org/10.17504/protocols.io.bfshjnb6 Copy
Authors: Franchesca Farris, Marda Jorgensen
Group: Human BioMolecular Atlas Program (HuBMAP) Method Development Community
Summary: This is an appendix for spleen SOP.
Proper citation: Franchesca Farris, Marda Jorgensen 2020. SOP Appendix for Spleen. protocols.io dx.doi.org/10.17504/protocols.io.bcwhixb6 Copy
Authors: Liz McDonough, Chrystal Chadwick, Fiona Ginty, Christine Surrette, Anup Sood
Group: Human BioMolecular Atlas Program (HuBMAP) Method Development Community, GE Research
Summary: The purpose of this protocol is to manually deparaffinize and rehydrate slides for the Cell DIVE™ Platform.
Proper citation: Liz McDonough, Chrystal Chadwick, Fiona Ginty, Christine Surrette, Anup Sood 2020. Cell DIVE™ Platform | Slide Clearing and Antigen Retrieval. protocols.io dx.doi.org/10.17504/protocols.io.bpwumpew Copy
Authors: Maigan Brusko
Group: Human BioMolecular Atlas Program (HuBMAP) Method Development Community
Summary: This standard operating procedure (SOP) provides instructions for staining and sorting live cells. This SOP applies to cryopreserved cells that are stained with Live/Dead and sorted in the BD ARIA III to remove dead cells prior to 10x sequencing.
Proper citation: Maigan Brusko 2020. HuBMAP UF TMC - FACS Sorting of Live Cells for 10x scRNASeq. protocols.io dx.doi.org/10.17504/protocols.io.bfqzjmx6 Copy
Authors: Aaron Horning
Group: NCIHTAN, Human BioMolecular Atlas Program (HuBMAP) Method Development Community
Proper citation: Aaron Horning 2020. Storage and Processing of Tissue for bulk RNA Isolation. protocols.io dx.doi.org/10.17504/protocols.io.bf52jq8e Copy
Authors: Marda Jorgensen, Michelle Daniel
Group: Human BioMolecular Atlas Program (HuBMAP) Method Development Community
Summary: Version 11 of the antibody labelling protocol published by Fluidigm.
Proper citation: Marda Jorgensen, Michelle Daniel 2020. Maxpar Antibody Labeling for Imaging Mass Cytometry. protocols.io dx.doi.org/10.17504/protocols.io.bf5jjq4n Copy
Authors: Danielle Gutierrez, Jamie Allen, Zach Jenkins, Jeff Spraggins
Group: VU Biomolecular Multimodal Imaging Center, Human BioMolecular Atlas Program (HuBMAP) Method Development Community
Summary: Description of settings used to acquire LC-MS/MS data from label-free proteomic samples.
Proper citation: Danielle Gutierrez, Jamie Allen, Zach Jenkins, Jeff Spraggins 2021. LC-MS/MS Label-Free Proteomic Data Acquisition. protocols.io dx.doi.org/10.17504/protocols.io.bs7hnhj6 Copy
Authors: Jamie Allen, Elizabeth Neumann, Maya Brewer, Jeff Spraggins, Danielle Gutierrez, Mark De Caestecker
Group: VU Biomolecular Multimodal Imaging Center, Human BioMolecular Atlas Program (HuBMAP) Method Development Community
Summary: Scope: Extract RNA for RIN and DV 200 Assessment.Expected Outcome: RIN and DV 200 measurements for tissue quality assessment.
Proper citation: Jamie Allen, Elizabeth Neumann, Maya Brewer, Jeff Spraggins, Danielle Gutierrez, Mark De Caestecker 2020. RNA Extraction for RIN and DV 200 Analysis. protocols.io dx.doi.org/10.17504/protocols.io.86nhzde Copy
Authors: Elizabeth Neumann, Jamie Allen, Maya Brewer, David Anderson, Mark De Caestecker, Danielle Gutierrez, Jeff Spraggins
Group: VU Biomolecular Multimodal Imaging Center, Human BioMolecular Atlas Program (HuBMAP) Method Development Community
Summary: We aim to develop high resolution, chemically informative imaginig methodologies for building an atlas of human organs, such as the kidney.Scope:Provide an overview of the methods used by the Vanderbilt Tissue Mapping Center as part of the Human Biomolecular Atlas Program (HuBMAP, NIH Common Fund) and contextualize individual protocols within our larger workflow.
Proper citation: Elizabeth Neumann, Jamie Allen, Maya Brewer, David Anderson, Mark De Caestecker, Danielle Gutierrez, Jeff Spraggins 2020. VU Biomolecular Multimodal Imaging Center (BIOMIC) kidney characterization pipeline for tissues collected through the Cooperative Human Tissue Network (CHTN). protocols.io dx.doi.org/10.17504/protocols.io.beevjbe6 Copy
Authors: David Anderson, Elizabeth Neumann, Jamie Allen, Maya Brewer, Danielle Gutierrez, Jeff Spraggins
Group: VU Biomolecular Multimodal Imaging Center, Human BioMolecular Atlas Program (HuBMAP) Method Development Community
Summary: Scope: Protocol for sectioning flash frozen tissue that can be used for 3D IMS or MxIF experiments.Expected Outcome:Serial sections from a tissue that IMS and MxIF can be performed on with subsequent 3D reconstruction
Proper citation: David Anderson, Elizabeth Neumann, Jamie Allen, Maya Brewer, Danielle Gutierrez, Jeff Spraggins 2019. Cryostat Sectioning of Tissues for 3D Multimodal Molecular Imaging. protocols.io dx.doi.org/10.17504/protocols.io.7ethjen Copy
Authors: Marda Jorgensen, Seth Currlin
Group: Optical Clearing of Tissue, Human BioMolecular Atlas Program (HuBMAP) Method Development Community
Summary: The Zeiss Lightsheet 7 is used to quickly and efficiently scan large tissue volumes using laser lightsheet illumination. The focused laser sheets and imaging plane are static while the tissue is moved throughout the selected volume for three dimensional image acquisition. This protocol will cover the procedures used for the day of lightsheet image acquisition.Setting the acquisition volume: Large scanning volumes are composed of multiple z-stack being stitched back together based on overlapping edges of the scanning window. This requires setting an overall z-stack, which must include the entirety of tissue as covered by adjoining z-stacks. This becomes tricky when considering irregularly shaped tissues and their orientation relative to the various laser and imaging objectives.Dual-side lightsheet alignment: To obtain a strong and clear image from large tissues it is helpful to illuminate the sample with both the left and right laser. Each lasersheet can be angled slightly within the focal plane to account for differences in the R.I. of the tissue and R.I. media (TDE). This is a manual tuning process and is not intended for major R.I. differences.Protocol Overview:- Refractive index matching of cleared tissue samples. - Mounting tissue onto sample holder.- Image acquisition in Zen 3.1 LS (black edition), including: - Setting the acquisition volume - Dual-sided lightsheet alignment
Proper citation: Marda Jorgensen, Seth Currlin 2020. Lightsheet Microscope Acquisition Protocol. protocols.io dx.doi.org/10.17504/protocols.io.begajbse Copy
Authors: Jerelyn Nick, Marda Jorgensen, Seth Currlin
Group: Human BioMolecular Atlas Program (HuBMAP) Method Development Community
Summary: This protocol is an overview of all of the protocols currently in use for the Light Sheet Fluorescence Microscopy modality at HubMAP Tissue Mapping Center,TMC-Florida/Zurich. It includes protocols.io links to each of the individual protocols that make up this project workflow, from processing tissue and imaging the samples, to uploading the final data to the HuBMAP HIVE.
Proper citation: Jerelyn Nick, Marda Jorgensen, Seth Currlin 2020. HuBMAP TMC-Florida/Zurich Light Sheet Fluorescence Microscopy Modality Overview. protocols.io dx.doi.org/10.17504/protocols.io.bqaamsae Copy
Authors: Blue Lake, Elizabeth Duong, Dinh Diep, Xin Sun, Peter K, Jim Hagood, Gloria Pryhuber, Sanjay Jain, Kun Zhang [University Of California
Group: Human BioMolecular Atlas Program (HuBMAP) Method Development Community
Summary: This document provides an overview of the protocols used by the Human Kidney, Urinary Tract, and Lung Mapping Center (KULMAP) for generation of spatially resolved mulitomic cell type organ maps within HuBMAP. This involves sequencing of the transcriptomes and epigenomes of dissociated single cells in a massively parallel manner, which will then inform on a highly multiplexed RNA in situ imaging method (DART-FISH) for spatial mapping of hundreds of molecular targets in the tissue sections, at a subcellular resolution. These spatial molecular maps will serve as scaffolds for computational registration of cell types and the associated transcriptome/chromatin maps to the tissue space.
Proper citation: Blue Lake, Elizabeth Duong, Dinh Diep, Xin Sun, Peter K, Jim Hagood, Gloria Pryhuber, Sanjay Jain, Kun Zhang [University Of California 2020. Human Kidney, Urinary Tract, and Lung Cell Type Mapping Pipeline for the Human Biomolecular Atlas Program (HuBMAP). protocols.io dx.doi.org/10.17504/protocols.io.bj9wkr7e Copy
Authors: Elizabeth Neumann, Jamie Allen, Maya Brewer, David Anderson, Mark De Caestecker, Danielle Gutierrez, Jeff Spraggins
Group: VU Biomolecular Multimodal Imaging Center, Human BioMolecular Atlas Program (HuBMAP) Method Development Community
Summary: We aim to develop high resolution, chemically informative imaginig methodologies for building an atlas of human organs, such as the kidney.Scope:Provide an overview of the methods used by the Vanderbilt Tissue Mapping Center as part of the Human Biomolecular Atlas Program (HuBMAP, NIH Common Fund) and contextualize individual protocols within our larger workflow.
Proper citation: Elizabeth Neumann, Jamie Allen, Maya Brewer, David Anderson, Mark De Caestecker, Danielle Gutierrez, Jeff Spraggins 2020. VU Biomolecular Multimodal Imaging Center (BIOMIC) kidney characterization pipeline for tissues collected through the Cooperative Human Tissue Network (CHTN). protocols.io dx.doi.org/10.17504/protocols.io.bfskjncw Copy
Authors: Ho, Jonhan
Group: Human BioMolecular Atlas Program (HuBMAP) Method Development Community, GE Research
Summary: This document outlines the required criteria for donor inclusion of skin specimens in the Human BioMolecular Atlas Program (HuBMAP). The study intends to be as inclusive as possible of race and ethnicity, dependent only on the consent for donor tissue for research. Disease known at the time of donation or later identified during the research will not necessarily exclude the sample from the research program.
Proper citation: Ho, Jonhan 2020. Donor Acceptance Criteria for GE/UPitt HuBMAP Inclusion. protocols.io dx.doi.org/10.17504/protocols.io.bqi6muhe Copy
Authors: Michelle Daniel, Marda Jorgensen
Group: Human BioMolecular Atlas Program (HuBMAP) Method Development Community
Summary: This SOP describes the preparation of compensation slides with single antibodyconjugatespots that can be used to estimate mass channel spillover for IMC. Spillovervaries from metal lot to lot due to varying impurities. Once a spillover matrix for a specificset of antibody-conjugates has been generated, the experiment of interest, in which thesame antibody conjugates have been used, can be spillover-corrected.It is crucial that this procedure is performed for the exact same antibody conjugates thatare used to pipette the antibody panel for sample staining.The original publication can be viewed here:“Compensation of Signal Spillover in Suspension and Imaging Mass Cytometry” by S.Chevrier, HL Crowell and VRT Zanotelli et al., Cell Systems, 2018
Proper citation: Michelle Daniel, Marda Jorgensen 2020. Imaging Mass Cytometry Compensation Slide Preparation. protocols.io dx.doi.org/10.17504/protocols.io.bf2djqa6 Copy
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