KPMP KIDNEY TISSUE ATLAS documentation

JUMP TO:

• ‍Regional transcriptomics
• ‍Single-cell RNA-seq‍
• ‍Single-nucleus RNA-seq‍
• Single-nucleus (sn) RNA-seq + snATAC-seq (10X Multiome)
• ‍Spatial transcriptomics

Single-nucleus RNA-seq

Goal:
Identify cell types and states associated with normal and injured kidney functions using gene expression profiling. Identify marker genes for cell type/states and any proportion shifts underlying pathology.

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Protocols:

• Preparation
• Isolation of single nuclei from solid tissues
• snDrop-seq
• 10x Genomics snRNA sequequencing for TRA

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Metadata standards

• Single-nucleus metadata template

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Single-cell RNA-seq

Goal:
Empirically derive cell subtypes and cell-type-specific gene expression profiles​.

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Protocol(s)

• Total RNA isolation from bulk tissue or isolated cells
• Single-cell RNA sequencing

Metadata standards

• Single-cell metadata template

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Regional transcriptomics

Goal:
Generate deep transcriptomic signatures from nephron segments defined spatially by antibody staining using laser microdissection.

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Protocol(s)

• Cryopreservation
• Laser microdissection for regional transcriptomics

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Metadata standards

• Regional transcriptomics metadata template
• Bulk transcriptomics metadata template

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Spatial transcriptomics

Goal:
Capture whole transcriptome mRNA expression with localization to kidney cells and structures.

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Protocol(s)

• Spatial transcriptomics protocol

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Metadata standards

• Not yet available

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Single-nucleus (sn) RNA-seq + snATAC-seq (10X Multiome)

Goal:
10X Genomics multiome protocol generates a molecular atlas of the human kidney with comprehensive cell types and minimal processing artifacts. 10X Multiome snATAC-seq + Gene Expression assay allows measurements of chromatin accessibility and transcription from the same cell. This allows direct insights into putative transcription factors or SNPs associated with open chromatin and impact on gene expression.

PROTOCOLS:

• ‍10X Genomics Single-Nucleus Multiome (RNA + ATAC) Assay for Profiling Adult Human Tissues V.2 

METADATA STANDARDS

• 10X Multiome metadata template

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JUMP TO:

• Regional proteomics

Regional proteomics

Goal:
Characterize the kidney proteome in health and disease to identify protein markers that reflect each segment, matrix, and cell type contained in the tissue.

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Protocol(s)

• Laser microdissection (LMD) for regional proteomics‍

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Metadata standards

• Not yet available

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JUMP TO:

• ‍Spatial metabolomics
  
• Spatial lipidomics
• Spatial N-glycomics
  

Spatial metabolomics

Goal:
Localize the small metabolite markers in tissue sections from human kidneys.

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Protocol(s)

• Matrix-Assisted Laser Desorption/Ionization-Mass Spectrometric Imaging (MALDI-MSI)
• Tissue Preparation for Spatial Metabolomics
• Optical Image Collection

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Metadata standards

• Spatial metabolomics metadata template

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Spatial lipidomics

Goal:
Localize the lipid markers in tissue sections from human  kidneys.

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Protocol(s)

• Matrix-Assisted Laser Desorption/Ionization-Mass Spectrometric Imaging (MALDI-MSI)

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Metadata standards

• Spatial lipidomics metadata template

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Spatial N-glycomics

Goal:
Localize the N-glycan markers in tissue sections from human  kidneys

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Protocol(s)

• Matrix-Assisted Laser Desorption/Ionization-Mass Spectrometric Imaging (MALDI-MSI)

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Metadata standards

• Spatial N-glycomics metadata template

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JUMP TO:

• Cut & Run
• ‍DNA methyl-seq
  

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CUT&RUN

Goal:

To obtain genome-wide profiles of histone post-translational modifications that define chromatin states of healthy and injured/repairing kidney tissue using Cleavage Under Targets and Release Using Nuclease (CUT&RUN). This technology enables the interrogation of chromatin profiles with limited tissue, such as kidney biopsies. 

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PROTOCOLS:

• CUT&RUN Chromatin Profiling of Human Kidney Tissue

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METADATA STANDARDS

• CUT&RUN template

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DNA METHYLATION SEQUENCING

Goal:

The goal of this technology is to uncover methylation signatures of kidney sub-segments that are key determinants of kidney disease progression. This work will result in a comprehensive set of methylation patterns and genotypes to map back to a kidney atlas. This will allow the characterization of methylation signatures of over 20 million CpG sites in individual kidney compartments from both nephrectomies and biopsies using laser microdissection (LMD) methodology with downstream whole-genome bisulfite-sequencing (WGBS).

Workflow: 1) Cryosectioning, 2) LMD processing, 3) DNA isolation, bisulfate conversion, WGBS-sequencing, 4) aligning with the reference genome with Bismark, 5) data analysis in R, and genome program visualization, 6) raw and analyzed data are uploaded to the KPMP data lake.

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PROTOCOLS:

• ‍Laser microdissection for regional transcriptomics and proteomics V.2

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METADATA STANDARDS

• DNA methyl-seq template

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JUMP TO:

• 3D tissue imaging and cytometry
• CODEX

3D tissue imaging and cytometry

Goal:
Understand the kidney in health and disease via 3D imaging and cytometric analyses.

The following image types within this technology are available in the Kidney Tissue Atlas:

• Composite 3D 8-channel immunofluorescence image volume
  3D volume completely represented as a stack of individual, 8-channel images. Every focal plane image and every channel can be independently inspected.
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• Composite max projection of 8-channel immunofluorescence image volume
  8-channel volume combined into a single maximum projection; composite image consists of 8 channels.
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• RGB max projection of 2-channel (autofluorescence and second harmonic generation) image volume
  Projection of 3D volume collected prior to labeling; channels cannot be controlled.
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• RGB max projection of 8-channel immunofluorescence image volume
  8-channel volume combined into a single maximum projection and converted to RGB color space.

Protocol(s)

• Sample preparation and imaging for large scale 3D spectral confocal imaging of tissues

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Metadata standards

• Not yet available

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CODEX (CO-detection by InDEXing)

Goal:

To build a library of high-resolution phenotypical maps of kidney biopsies with anchor, immune, and functional markers for in situ spatial analysis at single cell resolution in normal and pathological conditions.

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Protocol(s)

• Not yet available

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Metadata standards

• Not yet available

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