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10X Genomics Single-Nucleus RNA-Sequencing for Transcriptomic Profiling of Adult Human Tissues Resource Report Resource Website |
Sarah Urata, Blue Lake, Dinh Diep, Masato Hoshi, Sanjay Jain, Kun Zhang | DOI:10.17504/protocols.io.86khzcw | KPMP, Human BioMolecular Atlas Program (HuBMAP) Method Development Community | 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. | University of California, San Diego, University of California, San Diego, University of California, San Diego, Washington University, Saint Louis, Washington University School of Medicine, Saint Louis, University of California, San Diego | http://genome-tech.ucsd.edu/ZhangLab/ | 3 | 2019 | 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 https://dx.doi.org/10.17504/protocols.io.86khzcw | 2021-04-15 09:14:25 | ||
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LC-MS/MS Label-Free Proteomic Data Analysis Parameters Resource Report Resource Website |
Danielle Gutierrez, Jamie Allen, Zach Jenkins, Jeff Spraggins | DOI:10.17504/protocols.io.bfshjnb6 | VU Biomolecular Multimodal Imaging Center, Human BioMolecular Atlas Program (HuBMAP) Method Development Community | List of parameters and settings for searching label free proteomic data in MaxQuant version 1.6.7. | Vanderbilt University, Vanderbilt University, Vanderbilt University, Vanderbilt University | 2 | 2020 | Danielle Gutierrez, Jamie Allen, Zach Jenkins, Jeff Spraggins 2020. LC-MS/MS Label-Free Proteomic Data Analysis Parameters. protocols.io https://dx.doi.org/10.17504/protocols.io.bfshjnb6 | 2021-04-15 09:14:26 | |||
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Cell DIVE™ Platform | Slide Clearing and Antigen Retrieval Resource Report Resource Website |
Liz McDonough, Chrystal Chadwick, Fiona Ginty, Christine Surrette, Anup Sood | DOI:10.17504/protocols.io.bpwumpew | Human BioMolecular Atlas Program (HuBMAP) Method Development Community, GE Research | The purpose of this protocol is to manually deparaffinize and rehydrate slides for the Cell DIVE™ Platform. | GE Research, GE Research, GE Research, GE Research, GE Research | 1 | 2020 | Liz McDonough, Chrystal Chadwick, Fiona Ginty, Christine Surrette, Anup Sood 2020. Cell DIVE™ Platform | Slide Clearing and Antigen Retrieval. protocols.io https://dx.doi.org/10.17504/protocols.io.bpwumpew | 2021-04-15 09:14:33 | |||
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HuBMAP UF TMC - FACS Sorting of Live Cells for 10x scRNASeq Resource Report Resource Website |
Maigan Brusko | DOI:10.17504/protocols.io.bfqzjmx6 | Human BioMolecular Atlas Program (HuBMAP) Method Development Community | 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. | University of Florida | 1 | 2020 | Maigan Brusko 2020. HuBMAP UF TMC - FACS Sorting of Live Cells for 10x scRNASeq. protocols.io https://dx.doi.org/10.17504/protocols.io.bfqzjmx6 | 2021-04-15 09:14:36 | |||
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SOP Appendix for Spleen Resource Report Resource Website |
Franchesca Farris, Marda Jorgensen | DOI:10.17504/protocols.io.bcwhixb6 | Human BioMolecular Atlas Program (HuBMAP) Method Development Community | This is an appendix for spleen SOP. | University of Florida, University of Florida | 2 | 2020 | Franchesca Farris, Marda Jorgensen 2020. SOP Appendix for Spleen. protocols.io https://dx.doi.org/10.17504/protocols.io.bcwhixb6 | 2021-04-15 09:14:31 | |||
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VU Biomolecular Multimodal Imaging Center (BIOMIC) kidney characterization pipeline for tissues collected through the Cooperative Human Tissue Network (CHTN) Resource Report Resource Website |
Elizabeth Neumann, Jamie Allen, Maya Brewer, David Anderson, Mark De Caestecker, Danielle Gutierrez, Jeff Spraggins | DOI:10.17504/protocols.io.7hhhj36 | VU Biomolecular Multimodal Imaging Center, Human BioMolecular Atlas Program (HuBMAP) Method Development Community | 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. | Vanderbilt University, Vanderbilt University, Vanderbilt University, Vanderbilt University, Division of Nephrology, Vanderbilt University Medical Center, Vanderbilt University, Vanderbilt University | 1 | 2019 | Elizabeth Neumann, Jamie Allen, Maya Brewer, David Anderson, Mark De Caestecker, Danielle Gutierrez, Jeff Spraggins 2019. VU Biomolecular Multimodal Imaging Center (BIOMIC) kidney characterization pipeline for tissues collected through the Cooperative Human Tissue Network (CHTN). protocols.io https://dx.doi.org/10.17504/protocols.io.7hhhj36 | 2021-04-15 09:14:32 | |||
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Maxpar Antibody Labeling for Imaging Mass Cytometry Resource Report Resource Website |
Marda Jorgensen, Michelle Daniel | DOI:10.17504/protocols.io.bf5jjq4n | Human BioMolecular Atlas Program (HuBMAP) Method Development Community | Version 11 of the antibody labelling protocol published by Fluidigm. | University of Florida, University of Zürich | 1 | 2020 | Marda Jorgensen, Michelle Daniel 2020. Maxpar Antibody Labeling for Imaging Mass Cytometry. protocols.io https://dx.doi.org/10.17504/protocols.io.bf5jjq4n | 2021-04-15 09:14:51 | |||
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RNA Extraction for RIN and DV 200 Analysis Resource Report Resource Website |
Jamie Allen, Elizabeth Neumann, Maya Brewer, Jeff Spraggins, Danielle Gutierrez, Mark De Caestecker | DOI:10.17504/protocols.io.86nhzde | VU Biomolecular Multimodal Imaging Center, Human BioMolecular Atlas Program (HuBMAP) Method Development Community | Scope: Extract RNA for RIN and DV 200 Assessment.Expected Outcome: RIN and DV 200 measurements for tissue quality assessment. | Vanderbilt University, Vanderbilt University, Vanderbilt University, Vanderbilt University, Vanderbilt University, Division of Nephrology, Vanderbilt University Medical Center | 1 | 2020 | Jamie Allen, Elizabeth Neumann, Maya Brewer, Jeff Spraggins, Danielle Gutierrez, Mark De Caestecker 2020. RNA Extraction for RIN and DV 200 Analysis. protocols.io https://dx.doi.org/10.17504/protocols.io.86nhzde | 2021-04-15 09:14:52 | |||
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FFPE Tissue Sectioning for CODEX Resource Report Resource Website |
Marda Jorgensen, Franchesca Farris | DOI:10.17504/protocols.io.8mfhu3n | Human BioMolecular Atlas Program (HuBMAP) Method Development Community | FFPE tissues for CODEX® analysis must adhere directly onto Poly-lysine-coated coverslips. Preparation and storage of tissue slices are critical for sample integrity. | University of Florida, University of Florida | 1 | 2019 | Marda Jorgensen, Franchesca Farris 2019. FFPE Tissue Sectioning for CODEX. protocols.io https://dx.doi.org/10.17504/protocols.io.8mfhu3n | 2021-04-15 09:14:45 | |||
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SOP Appendix for Thymus Resource Report Resource Website |
Franchesca Farris | DOI:10.17504/protocols.io.bbgkijuw | Human BioMolecular Atlas Program (HuBMAP) Method Development Community | This is the appendix for the Thymus SOP. | University of Florida | 1 | 2020 | Franchesca Farris 2020. SOP Appendix for Thymus . protocols.io https://dx.doi.org/10.17504/protocols.io.bbgkijuw | 2021-04-15 09:14:48 | |||
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seqFISH Hyb Station Resource Report Resource Website |
Long Cai, Linus Eng, Nico Pierson, Chris Cronin | DOI:10.17504/protocols.io.bhfkj3kw | Human BioMolecular Atlas Program (HuBMAP) Method Development Community | The seqFISH Hyb Station is a custom built platform consists of a liquid handling pump , a fluidic switcher , and a 96-well plate auto-sampler which aids seqFISH experiments by flowing hybridization reagents and washes in and out of a flow cell containing the biological sample. Researchers set up multi-day experiments to perform chemistries such as hybridizations, washes, and stripping. In each round of hybridization, the auto sampler handle will move to the designated well and approximately 100uL of readouts reagents are flowed into the custom made flow cell to allow hybridization. Following by imaging with a fluorescent microscope, the signals are extinguished by flowing in excess high formamide solution to melt away the readout probes. These steps are repeated until all hybridization rounds are completed. The controls of seqFISH Hyb Station and imaging are controlled and integrated using the open source software Micro-Manager. | California Institute of Technology, California Institute of Technology, California Institute of Technology, California Institute of Technology | 1 | 2020 | Long Cai, Linus Eng, Nico Pierson, Chris Cronin 2020. seqFISH Hyb Station. protocols.io https://dx.doi.org/10.17504/protocols.io.bhfkj3kw | 2021-04-15 09:14:48 | |||
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Donor Acceptance Criteria for GE/UPitt HuBMAP Inclusion Resource Report Resource Website |
Ho, Jonhan | DOI:10.17504/protocols.io.bqi6muhe | Human BioMolecular Atlas Program (HuBMAP) Method Development Community, GE Research | 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. | University of Pittsburgh Medical Center | 1 | 2020 | Ho, Jonhan 2020. Donor Acceptance Criteria for GE/UPitt HuBMAP Inclusion. protocols.io https://dx.doi.org/10.17504/protocols.io.bqi6muhe | 2021-04-15 09:15:23 | |||
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Imaging Mass Cytometry Modality Overview Resource Report Resource Website |
Michelle Daniel, Marda Jorgensen | DOI:10.17504/protocols.io.bf3kjqkw | Human BioMolecular Atlas Program (HuBMAP) Method Development Community | Unversity of Zurich, University of Florida | 1 | 2020 | Michelle Daniel, Marda Jorgensen 2020. Imaging Mass Cytometry Modality Overview . protocols.io https://dx.doi.org/10.17504/protocols.io.bf3kjqkw | 2021-04-15 09:14:56 | ||||
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Imaging Mass Cytometry Compensation Slide Preparation Resource Report Resource Website |
Michelle Daniel, Marda Jorgensen | DOI:10.17504/protocols.io.bf2djqa6 | Human BioMolecular Atlas Program (HuBMAP) Method Development Community | 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 | University of Zürich, University of Florida | 1 | 2020 | Michelle Daniel, Marda Jorgensen 2020. Imaging Mass Cytometry Compensation Slide Preparation. protocols.io https://dx.doi.org/10.17504/protocols.io.bf2djqa6 | 2021-04-15 09:15:30 | |||
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LC-MS/MS Label-Free Proteomic Data Acquisition Resource Report Resource Website |
Danielle Gutierrez, Jamie Allen, Zach Jenkins, Jeff Spraggins | DOI:10.17504/protocols.io.bft3jnqn | VU Biomolecular Multimodal Imaging Center, Human BioMolecular Atlas Program (HuBMAP) Method Development Community | Description of settings used to acquire LC-MS/MS data from label-free proteomic samples. | Vanderbilt University, Vanderbilt University, Vanderbilt University, Vanderbilt University | 3 | 2020 | Danielle Gutierrez, Jamie Allen, Zach Jenkins, Jeff Spraggins 2020. LC-MS/MS Label-Free Proteomic Data Acquisition. protocols.io https://dx.doi.org/10.17504/protocols.io.bft3jnqn | 2021-04-15 09:15:32 | |||
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Data Processing and Preparation of MALDI IMS data Resource Report Resource Website |
Nathan Heath Patterson, Elizabeth Neumann, Jamie Allen, Danielle Gutierrez, Jeff Spraggins | DOI:10.17504/protocols.io.bed3ja8n | VU Biomolecular Multimodal Imaging Center, Human BioMolecular Atlas Program (HuBMAP) Method Development Community | Scope:How to process a mass spectrometry imaging data into a final imzML file. | Vanderbilt University, Vanderbilt University, Vanderbilt University, Vanderbilt University, Vanderbilt University | 1 | 2020 | Nathan Heath Patterson, Elizabeth Neumann, Jamie Allen, Danielle Gutierrez, Jeff Spraggins 2020. Data Processing and Preparation of MALDI IMS data. protocols.io https://dx.doi.org/10.17504/protocols.io.bed3ja8n | 2021-04-15 09:15:31 | |||
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HuBMAP: Embedding Fresh Frozen OCT Samples Resource Report Resource Website |
Marda Jorgensen, Jerelyn Nick | DOI:10.17504/protocols.io.bcwsixee | Human BioMolecular Atlas Program (HuBMAP) Method Development Community | The purpose of this Standard Operating Procedure (SOP) is to outline procedures for the OCT embedding of HuBMAP fresh frozen specimens. | university of Florida, University of Florida | 1 | 2020 | Marda Jorgensen, Jerelyn Nick 2020. HuBMAP: Embedding Fresh Frozen OCT Samples. protocols.io https://dx.doi.org/10.17504/protocols.io.bcwsixee | 2021-04-15 09:15:37 | |||
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CODEX - Poly-l-Lysine Cover-Slip Preparation Resource Report Resource Website |
Leigh Propper, Franchesca Farris, Marda Jorgensen | DOI:10.17504/protocols.io.baxyifpw | Human BioMolecular Atlas Program (HuBMAP) Method Development Community | Poly-Lysine Coverslip Preparation This protocol describes the process of creating Poly-lysine-coated coverslips that are required for mounting tissue sections for the CODEX® experiment workflow. | University of Florida, University of Florida, University of Florida | 3 | 2020 | Leigh Propper, Franchesca Farris, Marda Jorgensen 2020. CODEX - Poly-l-Lysine Cover-Slip Preparation. protocols.io https://dx.doi.org/10.17504/protocols.io.baxyifpw | 2021-04-15 09:15:37 | |||
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Appendix for Spleen SOP Resource Report Resource Website |
Marda Jorgensen, Jerelyn Nick | DOI:10.17504/protocols.io.bbcviiw6 | Human BioMolecular Atlas Program (HuBMAP) Method Development Community | This is an appendix diagram for spleen tissue cutting. | university of Florida, University of Florida | 1 | 2020 | Marda Jorgensen, Jerelyn Nick 2020. Appendix for Spleen SOP. protocols.io https://dx.doi.org/10.17504/protocols.io.bbcviiw6 | 2021-04-15 09:15:40 | |||
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HuBMAP: Embedding Fixed Frozen OCT Samples Resource Report Resource Website |
Marda Jorgensen, Jerelyn Nick | DOI:10.17504/protocols.io.basniede | Human BioMolecular Atlas Program (HuBMAP) Method Development Community | The purpose of this Standard Operating Procedure (SOP) is to outline procedures for the OCT embedding of HuBMAP frozen fixed specimens. | University of Florida, University of Florida | 1 | 2020 | Marda Jorgensen, Jerelyn Nick 2020. HuBMAP: Embedding Fixed Frozen OCT Samples. protocols.io https://dx.doi.org/10.17504/protocols.io.basniede | 2021-04-15 09:15:15 |
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