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Authors: Blue Lake, Kun Zhang
Group: Human Cell Atlas Method Development Community, KPMP, Human BioMolecular Atlas Program (HuBMAP) Method Development Community
Summary: Nuclei can be readily isolated from frozen tissues with a combination of chemical and physical treatments that can circumvent the non-uniform or incomplete dissociation of solid tissues into single cells. The isolation of nuclei can also circumvent RNA degradation or any introduction of technical artefacts (such as stress responses) that could be triggered during whole cell dissociation methods. Data generated from single-nucleus genomic assays permits discovery of molecular cell types that can be used to define the overall cellular makeup of a tissue or organ, and ultimately will inform upon adult human tissue atlases.
Proper citation: Blue Lake, Kun Zhang 2019. Isolation of single nuclei from solid tissues. protocols.io dx.doi.org/10.17504/protocols.io.ufketkw Copy
Authors: Michelle Daniel, Marda Jorgensen
Group: Human BioMolecular Atlas Program (HuBMAP) Method Development Community
Summary: This SOP describes the tuning of an Imaging Mass Cytometer (IMC). The tuning processencompasses parameters such as Interface alignment, gas flow rate optimizations, laserpower optimization and detector voltage optimization. All these factors aim at having amachine that performs equally over time.The machine needs to pass specifications listed in this SOP in order to be used for dataacquisition.Machines do not need to be tuned daily but at least twice per week and upon every startup.Additionally, the adjustment of the laser energy is described in this SOP, whichneeds to be performed every 3 weeks.
Proper citation: Michelle Daniel, Marda Jorgensen 2020. Imaging Mass Cytometer Tuning . protocols.io dx.doi.org/10.17504/protocols.io.bf2gjqbw Copy
Authors: Seth Currlin, Marda Jorgensen, Jerelyn Nick
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 light sheet 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 light sheet 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 light sheet 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 laser sheet 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 light sheet alignment
Proper citation: Seth Currlin, Marda Jorgensen, Jerelyn Nick 2020. Light Sheet Fluorescence Microscopy Image Acquisition. protocols.io dx.doi.org/10.17504/protocols.io.bprsmm6e Copy
Authors: Jamie Allen, Elizabeth Neumann, Maya Brewer, Haichun Yang, Jeff Spraggins, Danielle Gutierrez
Group: VU Biomolecular Multimodal Imaging Center, Human BioMolecular Atlas Program (HuBMAP) Method Development Community
Summary: Scope:Prepare formalin fixed tissue with freezing fresh tissue protocol (dx.doi.org/10.17504/protocols.io.6wghfbw). This protocol provides the steps to prepare formalin fixed human kidney tissue and perform histology assessment for normalcy.Expected Outcome:Pathological assessment of kidney tissue for use in LC-MS/MS and imaging.
Proper citation: Jamie Allen, Elizabeth Neumann, Maya Brewer, Haichun Yang, Jeff Spraggins, Danielle Gutierrez 2020. Initial Rapid Pathology Assessment of Kidney Tissue. protocols.io dx.doi.org/10.17504/protocols.io.9dph25n Copy
Authors: Jamie Allen, Jeff Spraggins, Danielle Gutierrez
Group: VU Biomolecular Multimodal Imaging Center, Human BioMolecular Atlas Program (HuBMAP) Method Development Community
Summary: Scope:To describe the procedure for the lysis, reduction/alkylation, trypsin digestion, and clean-up of tissue. Lysis will cover the lysing of tissue and protein concentration. Acetone precipitation will cover the precipitation of proteins. Digestion will cover the process for digesting 100 µg of protein using the Agilent AssayMap Bravo Robot and Promega Rapid Trypsin/LysC. The clean-up of the cells will cover the desalting process on the Agilent AssayMap Bravo and the subsequent preparation of the samples for LC/MS peptide analysis.Expected Outcome/Data:Cell samples lysed, digested, and desalted for analysis on MS instrument. Samples to be analyzed within one or two days of desalting.
Proper citation: Jamie Allen, Jeff Spraggins, Danielle Gutierrez 2019. Automated, Rapid Preparation of Tissue Sections for Proteomic Analysis. protocols.io dx.doi.org/10.17504/protocols.io.z3pf8mn Copy
Authors: Franchesca Farris, Marda Jorgensen
Group: Human BioMolecular Atlas Program (HuBMAP) Method Development Community
Summary: UF H&E Staining
Proper citation: Franchesca Farris, Marda Jorgensen 2019. UF H&E Staining. protocols.io dx.doi.org/10.17504/protocols.io.8jthunn Copy
Authors: Long Cai, Nina Dar, Yiing Lin, Shin Lin
Group: Human BioMolecular Atlas Program (HuBMAP) Method Development Community
Summary: Tissue Preparation for seqFISH
Proper citation: Long Cai, Nina Dar, Yiing Lin, Shin Lin 2019. seqFISH Tissue Preservation. protocols.io dx.doi.org/10.17504/protocols.io.7e7hjhn Copy
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 dx.doi.org/10.17504/protocols.io.8mfhu3n Copy
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 dx.doi.org/10.17504/protocols.io.bbgkijuw Copy
Authors: Franchesca Farris, Marda Jorgensen
Group: Human BioMolecular Atlas Program (HuBMAP) Method Development Community
Summary: This is the appendix for the Thymus SOP.
Proper citation: Franchesca Farris, Marda Jorgensen 2020. SOP Appendix for Thymus . protocols.io dx.doi.org/10.17504/protocols.io.bcwgixbw Copy
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 dx.doi.org/10.17504/protocols.io.bhfkj3kw Copy
Authors: Yiing Lin, Shin Lin
Group: Human BioMolecular Atlas Program (HuBMAP) Method Development Community
Proper citation: Yiing Lin, Shin Lin 2020. HuBMAP Tissue Preservation Protocol v2. protocols.io dx.doi.org/10.17504/protocols.io.bqm5mu86 Copy
Authors: Leigh Propper, Franchesca Farris, Marda Jorgensen
Group: Human BioMolecular Atlas Program (HuBMAP) Method Development Community
Summary: Poly-Lysine Coverslip Preparation This section describes the process of creating Poly-lysine-coated coverslips that are used for the tissue sections in the CODEX® experiment workflow.
Proper citation: Leigh Propper, Franchesca Farris, Marda Jorgensen 2019. CODEX - Poly-l-Lysine Cover-Slip Preparation. protocols.io dx.doi.org/10.17504/protocols.io.baeribd6 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 2019. 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.7hhhj36 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 2020. LC-MS/MS Label-Free Proteomic Data Acquisition. protocols.io dx.doi.org/10.17504/protocols.io.bft3jnqn Copy
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:To process to register MALDI IMS images to different types of microscopy images.
Proper citation: Nathan Heath Patterson, Elizabeth Neumann, Jamie Allen, Danielle Gutierrez, Jeff Spraggins 2020. Image Registration of MALDI IMS to Microscopy. protocols.io dx.doi.org/10.17504/protocols.io.bed2ja8e Copy
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 dx.doi.org/10.17504/protocols.io.baxyifpw Copy
Authors: Sanjay Jain, Masato Hoshi, Amanda Knoten, Diane Salamon
Group: KPMP, Human BioMolecular Atlas Program (HuBMAP) Method Development Community, Jain Lab
Summary: Critical to generating robust single cell or tissue gene expression data is ensuring that the source samples are procured and processed in a way that minimizes artifacts. This is especially critical for generating single cell/nucleus gene expression data from adult human tissue samples that are difficult to dissociate and might require prolonged manipulations that will introduce stress response signatures. We describe methods to: minimize these unwanted processing effects to enable interrogation of limited tissues, such as biopsies; allow histological validation and quality assurance and control of the samples; ensure compatibility with both single cell assays and orthogonal technologies. This involves preparing fresh frozen O.C.T. embedded tissue cryoblocks to collect thick sections for gene expression studies and adjacent sections for QA/QC, bulk RNAseq, DNA preparation or multiplex in situ RNA hybridization. The protocol for embedding limited amount of tissue for cryosections and susequent OMICS studies should be applicable to several solid organs and biopsies.
Proper citation: Sanjay Jain, Masato Hoshi, Amanda Knoten, Diane Salamon 2019. Preparation of Adult Human Kidney Tissue for Single Nucleus RNA-seq and Other Multiomics Studies. protocols.io dx.doi.org/10.17504/protocols.io.568g9hw Copy
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 dx.doi.org/10.17504/protocols.io.bf3kjqkw Copy
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 dx.doi.org/10.17504/protocols.io.basniede Copy
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