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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 https://dx.doi.org/10.17504/protocols.io.86khzcw Copy   

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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 https://dx.doi.org/10.17504/protocols.io.bfshjnb6 Copy   

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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 https://dx.doi.org/10.17504/protocols.io.bpwumpew Copy   

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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 https://dx.doi.org/10.17504/protocols.io.bfqzjmx6 Copy   

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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 https://dx.doi.org/10.17504/protocols.io.bcwhixb6 Copy   

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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 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 Copy   

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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 https://dx.doi.org/10.17504/protocols.io.bf5jjq4n Copy   

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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 https://dx.doi.org/10.17504/protocols.io.86nhzde Copy   

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Authors: Marda Jorgensen, Franchesca Farris
Group: Human BioMolecular Atlas Program (HuBMAP) Method Development Community
Summary: FFPE tissues for CODEX® analysis must adhere directly onto Poly-lysine-coated coverslips. Preparation and storage of tissue slices are critical for sample integrity.

Proper citation: Marda Jorgensen, Franchesca Farris 2019. FFPE Tissue Sectioning for CODEX. protocols.io https://dx.doi.org/10.17504/protocols.io.8mfhu3n Copy   

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Authors: Franchesca Farris
Group: Human BioMolecular Atlas Program (HuBMAP) Method Development Community
Summary: This is the appendix for the Thymus SOP.

Proper citation: Franchesca Farris 2020. SOP Appendix for Thymus . protocols.io https://dx.doi.org/10.17504/protocols.io.bbgkijuw Copy   

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Authors: Long Cai, Linus Eng, Nico Pierson, Chris Cronin
Group: Human BioMolecular Atlas Program (HuBMAP) Method Development Community
Summary: 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.

Proper citation: Long Cai, Linus Eng, Nico Pierson, Chris Cronin 2020. seqFISH Hyb Station. protocols.io https://dx.doi.org/10.17504/protocols.io.bhfkj3kw Copy   

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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 https://dx.doi.org/10.17504/protocols.io.bqi6muhe Copy   

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Authors: Michelle Daniel, Marda Jorgensen
Group: Human BioMolecular Atlas Program (HuBMAP) Method Development Community

Proper citation: Michelle Daniel, Marda Jorgensen 2020. Imaging Mass Cytometry Modality Overview . protocols.io https://dx.doi.org/10.17504/protocols.io.bf3kjqkw Copy   

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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 https://dx.doi.org/10.17504/protocols.io.bf2djqa6 Copy   

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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 2020. LC-MS/MS Label-Free Proteomic Data Acquisition. protocols.io https://dx.doi.org/10.17504/protocols.io.bft3jnqn Copy   

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Authors: Nathan Heath Patterson, Elizabeth Neumann, Jamie Allen, Danielle Gutierrez, Jeff Spraggins
Group: VU Biomolecular Multimodal Imaging Center, Human BioMolecular Atlas Program (HuBMAP) Method Development Community
Summary: Scope:How to process a mass spectrometry imaging data into a final imzML file.

Proper citation: 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 Copy   

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Authors: Marda Jorgensen, Jerelyn Nick
Group: Human BioMolecular Atlas Program (HuBMAP) Method Development Community
Summary: The purpose of this Standard Operating Procedure (SOP) is to outline procedures for the OCT embedding of HuBMAP fresh frozen specimens.

Proper citation: Marda Jorgensen, Jerelyn Nick 2020. HuBMAP: Embedding Fresh Frozen OCT Samples. protocols.io https://dx.doi.org/10.17504/protocols.io.bcwsixee Copy   

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Authors: Leigh Propper, Franchesca Farris, Marda Jorgensen
Group: Human BioMolecular Atlas Program (HuBMAP) Method Development Community
Summary: 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.

Proper citation: 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 Copy   

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Authors: Marda Jorgensen, Jerelyn Nick
Group: Human BioMolecular Atlas Program (HuBMAP) Method Development Community
Summary: This is an appendix diagram for spleen tissue cutting.

Proper citation: Marda Jorgensen, Jerelyn Nick 2020. Appendix for Spleen SOP. protocols.io https://dx.doi.org/10.17504/protocols.io.bbcviiw6 Copy   

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Authors: Marda Jorgensen, Jerelyn Nick
Group: Human BioMolecular Atlas Program (HuBMAP) Method Development Community
Summary: The purpose of this Standard Operating Procedure (SOP) is to outline procedures for the OCT embedding of HuBMAP frozen fixed specimens.

Proper citation: Marda Jorgensen, Jerelyn Nick 2020. HuBMAP: Embedding Fixed Frozen OCT Samples. protocols.io https://dx.doi.org/10.17504/protocols.io.basniede Copy   

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