Single Cell Technology Core Services

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Let us help you design your next experiment! A well-controlled and balanced study will maximize information and minimize the overall cost. We offer consultations to help you decide the best platform and analysis strategy. Schedule a meeting with core director Dr. Mei Zhang, [email protected], to learn about all services and technologies available.

Supported Assays

  • Single Cell Gene Expression (3′ GEX)
  • Single Cell Immune Profiling (VDJ and 5′ GEX)
  • Single Cell Assay for Transposase Accessible Chromatin (ATAC)
  • Single Cell Multiome (3’GEX + ATAC)
  • Single Cell Fixed RNA Profiling
  • Single Cell long-read sequencing in collaboration with CHOP HTS core

Sample Requirements

Fresh samples:

Clean single cell or nuclei suspension free of debris and clumping

Cell Viability: >90% ideally, >70% is acceptable

Cell/nucleus concentration:

  • for targeting 500-10K cells: 700-1200 cells (or nuclei)/μl
  • for targeting 10K-20K cells: 1300-1600 cells (or nuclei)/μl

Minimum volume: 50 μl

Nuclei concentration requirement for ATAC seq depends on targeted recovery; please contact the core.

Preferred Buffers:

1X PBS (calcium and magnesium free) containing 0.04% weight/volume BSA (400 μg/ml).

PBS can be replaced with most common cell culture buffers and media if necessary. Media should not contain excessive amounts of EDTA (> 0.1mM) or magnesium (> 3mM), as those components will inhibit the reverse transcription reaction. Any surfactants (Tween-20, etc.) should also be avoided as they may interfere with GEM generation.

For nuclei: include 0.2U/μl RNase Inhibitor (Sigma Protector RNase inhibitor, PN-3335399001 is strongly recommended) in the buffer for gene expression or multiome (gene expression + ATAC) studies.

Fixed samples:

Samples need to be prepared with Chromium Next GEM Single Cell Fixed RNA Sample Preparation Kit (10x Genomics).

Number of cells required:

Singleplex:

  • Fixed cells 200,000-2 x 106
  • Fixed nuclei 400,000-2 x 106
  • FFPE dissociated suspension 400,000-2 x 106

Multiplex:

  • Fixed cells 50,000-2 x 106
  • Fixed nuclei 100,000-2 x 106
  • FFPE dissociated suspension 100,000-2 x 106

NanoString's GeoMx Digital Spatial Profiler (DSP)

Supported assays:

  • GeoMx Human Whole Transcriptome Atlas: 18,000+ human protein-coding genes
  • GeoMx Mouse Whole Transcriptome Atlas: 20,000+ mouse protein-coding genes
  • GeoMx Cancer Transcriptome Atlas: 1,800 human genes

Sample Requirements

  • Compatible samples: FFPE, Fresh or Fixed Frozen tissue
  • For best results, do not use FFPE blocks that are greater than 10 years old
  • It is essential to minimize RNases when processing fresh frozen tissues
  • Fisherbrand SuperFrost Plus slides or Leica BOND plus slides
  • Sectioning: FFPE 5 μm; Frozen 5-10 μm
  • Scan area (green area in the picture): 35.3mm x 14.1mm
  • Multiple sections can be mounted on the same slide, at least 2-3 mm apart

Vizgen MERSCOPE

Supported assays:

Custom designed gene panels for 140, 300, or 500 genes

Sample Requirements

  • Compatible samples: FFPE, Fresh or Fixed Frozen tissue
  • Maximum size: 1cm3
  • Sectioning: FFPE 4 or 5 μm; Frozen 10 μm
  • Mounted on MERSCOPE slides
  • RNA quality:
    • Frozen tissue: RIN>7 ideal; RIN 5-7: detection efficiency diminishes; RIN<5: do not proceed
    • FFPE: DV200>60% yield high quality data; DV200 40-60% yield quality data; DV200<40% yield low quality data

We strongly recommend testing sample quality with the MERSCOPE Sample Verification Kit.

10xGenomics Visium Spatial

Supported assays:

  • Human or mouse whole transcriptome gene expression
  • Human or mouse whole transcriptome gene expression and targeted protein expression (not available for HD)

Sample Requirements

  • Compatible samples: FFPE, Fresh or Fixed Frozen tissue; HD is only compatible with FFPE samples
  • Please ensure the desired tissue area will fit within the 6.5 x 6.5 mm or 11 x 11 mm capture area; only 6.5 x 6.5 mm is available for HD
  • There are two capture areas per slide
  • Tissue sections should be mounted within the allowable target area on the plain glass slide according to the 10x Genomics Tissue Preparation Guides
  • Recommended section thickness:
    • FFPE: 3-10 µm
    • FF or FxF: 10-20 µm
  • RNA quality of the block should be assessed prior to sectioning:
    • FFPE: DV200>30%
    • FF: RIN>4
    • FxF: DV200>50%

10xGenomics Xenium In Situ

Supported assays:

  • Human or mouse pre-designed panels
  • Standalone or add-on custom panels of up to 500 genes
  • Xenium Prime 5K, with optional add-on custom panels of up to 100 genes

Sample Requirements

  • Compatible samples: FFPE or Fresh Frozen tissue
  • Mounted on Xenium slides
  • Sample area: 10.45mm x 22.45mm
  • Section thickness: FFPE 5 μm; Frozen 10 μm
  • Prior to sectioning onto Xenium slides, tissue morphology should be checked for any undesired artifacts

NanoString's GeoMx Digital Spatial Profiler (DSP)

Supported assays

  • GeoMx protein assays for nCounter
  • GeoMx protein assays for NGS

Sample Requirements

“Sample
  • FFPE, Fresh or Fixed Frozen tissues
  • For best results, do not use FFPE blocks that are greater than 10 years old
  • Fisherbrand SuperFrost™ Plus slides or Apex BOND® slides
  • Sectioning: FFPE 5 μm; Frozen 5-10 μm
  • Tissue sections need to be mounted within the scan area (green area in the picture): 35.3mm x 14.1mm
  • Multiple sections can be mounted on the same slide, at least 2-3 mm apart

Akoya Phenocycler

Supported assays

  • Akoya inventoried antibodies for human fresh frozen and FFPE, and mouse fresh frozen
  • Custom conjugated antibodies

Sample Requirements

  • FFPE, Fresh or Fixed Frozen tissues
  • Coverslips (22mm x 22 mm, Akoyabio) coated with poly-L-lysine
  • Tissue sectioning: 5-10 μm (FFPE 4-5 μm, Frozen 8-10 μm); up to 15mm x 15mm

Our experienced bioinformaticians help with experimental design, developing reproducible workflows, analyzing high throughput next-generation sequencing data and spatial profiling data, and supporting manuscript development/publication. We generate visualizations of complex data and assist data uploads to public repositories.

Single Cell Services

  • CellRanger pipeline
  • Ambient RNA/background correction
  • QC analysis/filtering
  • Doublet detection
  • Merge/integration
  • Clustering
  • Summary with figures and commentary
  • Interactive data object
  • Additional offering – any published analysis
    • Cell type prediction
    • Custom reference genomes
    • Cluster analysis
    • Trajectory analysis
    • Pathway analysis

Spatial Biology Services

  • QC analysis/filtering
  • Merge/integration
  • Clustering
  • Summary with figures and commentary
  • Interactive data object
  • Additional offering – any published analysis
    • Interaction analysis
    • Cell type prediction
    • Neighbor enrichment
    • Differential spatial expression
    • Motif analysis
    • Spatial clustering