Top 3 Tips for Successful CosMx™ SMI Single-cell Spatial Runs at 1000 plex

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We are excited to share insights and recommendations to enhance your experience with CosMx™ SMI 1000-plex RNA assays. The quality of single-cell spatial data is influenced by various factors, and we aim to provide you with valuable information on experimental conditions for multiple tissue types to ensure the best results.  

CosMx 1000-plex RNA Assays: Considerations When Generating Single-Cell Spatial Data

Tip 1: Interpret Data by Tissue and Disease Type

Understanding the role of tissue/disease type is crucial for interpreting single-cell spatial data. We present mean number of transcripts (counts) detected per cell (Figure 1) and mean unique genes detected per cell (Figure 2) across various tissue/disease types. Data were collected from 210 externally-supplied tissue slides as part of the NanoString Technology Access Program (TAP), utilizing either the 1000-plex RNA Human Universal Cell Characterization (UCC) or the 1000-plex Mouse Neuroscience panel. Tissue samples presented are real-world samples that were not pre-curated for RNA quality upon receival to TAP.   

We make two key observations from this collated data: 1) RNA signal, as measured by transcripts/cell and unique genes/cell, varies greatly by tissue type. We attribute this trend, in part, to inherent tissue biology and panel gene content. 2) Tissue block and section quality are major factors for data quality, as evidenced by the signal heterogeneity observed within tissue types. We look forward to providing future guidance on improving sample quality prior to the CosMx workflow.

Tip 2: Consider Sample Preparation Conditions  

CosMx slide preparation protocol is in part defined by tissue-specific conditions. By utilizing the appropriate sample preparation and run conditions as well as contextualizing RNA signal by tissue type, users can better generate and interpret single-cell spatial data. For detailed guidance, please refer to the CosMx Slide Preparation Manual (Appendix I and II) and Sample Sectioning Tips and Tricks.  

In Table 1 below, we provide a summary of various tissue types and suggested modifications to experimental conditions that may be needed to optimize data quality. For the RNA Assay, digestion buffer concentration and incubation time for target retrieval may differ for some tissue types and will need to be empirically determined. We recommend starting with default conditions as per the CosMx Slide Preparation Manual and adjusting as required.  If tissue detachment is observed, begin slide preparation with a new tissue section and reduce target retrieval time. If the issue persists, consider reduction of the proteinase K concentration.   

Table 2 below breaks down target retrieval time, digestion conditions (concentration and time), and fiducial concentration for the various tissue types represented in Figure 1 and Figure 2 above. In addition, we provide recommended instrument pre-bleaching conditions for specific tissues. Refer to the CosMx SMI Instrument User Manual for detailed operational instructions. These conditions, empirically determined, may require user-specific adjustments. 

Tissue TypeTarget Retrieval TimeDigestion Buffer ConcentrationDigestion TimeFiducial ConcentrationPre-bleaching Profile
Appendix15 minutes3 µg/mL30 minutes0.0005%Configuration C
Bladder Cancer15 minutes3 µg/mL30 minutes0.0005%Configuration C
Bone Marrow15 minutes3 µg/mL30 minutes0.0010%Configuration C
Bone Marrow Cancer15 minutes3 µg/mL30 minutes0.0005%Configuration C
Brain15 minutes3 µg/mL30 minutes0.0005%Configuration B
Brain Alzheimer’s15 minutes3 µg/mL30 minutes0.0005%Configuration B
Brain Organoids8 minutes1 µg/mL15 minutes0.0005%Configuration C
Breast15 minutes3 µg/mL30 minutes0.0010%Configuration C
Breast Cancer15 minutes3 µg/mL30 minutes0.0010%Configuration C
Cartilage8 minutes3 µg/mL15 minutes0.0005%Configuration C
Cholangiocarcinoma15 minutes3 µg/mL30 minutes0.0005%Configuration C
Chronic Kidney Disease15 minutes3 µg/mL30 minutes0.0005%Configuration C
Colon15 minutes3 µg/mL30 minutes0.0010%Configuration C
Colitis15 minutes3 µg/mL30 minutes0.0005%Configuration C
Colon Cancer15 minutes3 µg/mL30 minutes0.0005%Configuration C
Glioblastoma15 minutes3 µg/mL30 minutes0.0005%Configuration C
Glioma15 minutes3 µg/mL30 minutes0.0005%Configuration C
Ileum15 minutes3 µg/mL30 minutes0.0005%Configuration C
Intestine15 minutes3 µg/mL30 minutes0.0005%Configuration C
Kidney15 minutes3 µg/mL30 minutes0.0010%Configuration B
Kidney Cancer15 minutes3 µg/mL30 minutes0.0010%Configuration B
Kidney Metabolic Disease15 minutes3 µg/mL30 minutes0.0010%Configuration B
Liver (normal)15 minutes3 µg/mL30 minutes0.0010%Configuration B
Liver (malignant)15 minutes3 µg/mL30 minutes0.0010%Configuration C
Lung15 minutes3 µg/mL30 minutes0.0005%Configuration C
Lung Cancer15 minutes3 µg/mL30 minutes0.0005%Configuration C
Lung Disease15 minutes3 µg/mL30 minutes0.0005%Configuration C
Lymph Node15 minutes3 µg/mL30 minutes0.0010%Configuration C
Lymphoma15 minutes3 µg/mL30 minutes0.0005%Configuration C
Mesothelioma15 minutes3 µg/mL30 minutes0.0005%Configuration C
Organoid8 minutes1 µg/mL30 minutes0.0005%Configuration C
Ovarian Cancer15 minutes3 µg/mL30 minutes0.0010%Configuration C
Pancreas Diabetes15 minutes3 µg/mL30 minutes0.0010%Configuration C
Pancreatic Cancer15 minutes3 µg/mL30 minutes0.0010%Configuration C
Pituitary Gland15 minutes3 µg/mL30 minutes0.0010%Configuration C
Placenta8 minutes1 µg/mL15 minutes0.0010%Configuration B
Placenta Diabetes15 minutes3 µg/mL30 minutes0.0005%Configuration B
Prostate Cancer15 minutes3 µg/mL30 minutes0.0010%Configuration C
Rectum15 minutes3 µg/mL30 minutes0.0005%Configuration C
Retina15 minutes3 µg/mL30 minutes0.0005%Configuration C
Skin15 minutes1 µg/mL30 minutes0.0010%Configuration C
Skin Cancer15 minutes3 µg/mL30 minutes0.0005%Configuration C
Skin Lupus8 minutes3 µg/mL 30 minutes0.0005%Configuration C
Stomach Cancer15 minutes3 µg/mL30 minutes0.0005%Configuration C
Tonsil15 minutes3 µg/mL30 minutes0.0005%Configuration C
Cell Pellet Array8 minutes1 µg/mL15 minutes0.0010%Configuration A
NHP Liver Infection15 minutes3 µg/mL30 minutes0.0005%Configuration C
NHP Lung Granuloma15 minutes3 µg/mL30 minutes0.0005%Configuration C
NHP Lymph Node SIV15 minutes3 µg/mL30 minutes0.0005%Configuration C
Mouse Artery15 minutes3 µg/mL30 minutes0.0010%Configuration C
Mouse Brain15 minutes3 µg/mL30 minutes0.0010%Configuration C
Mouse Brain Infection5 minutes3 µg/mL30 minutes0.0010%Configuration C
Mouse Brain Neurodegeneration15 minutes3 µg/mL30 minutes0.0010%Configuration C
Mouse Colon Infection15 minutes3 µg/mL30 minutes0.0010%Configuration C
Mouse Lung Infection8 minutes3 µg/mL*15 minutes0.0005%Configuration C
Mouse Retina15 minutes3 µg/mL30 minutes0.0005%Configuration C
Mouse Spleen HIV15 minutes3 µg/mL30 minutes0.0005%Configuration C

Tip 3: Plan Instrument Run Conditions

Instrument run time depends on the interrogated tissue area (number of Fields of View). Presented below (Table 3) are turnaround time estimations, based on different total FOV numbers per run. Sample integrity and data quality are influenced by instrument run time.  

For commercial software v1.2 at different total FOV numbers per run. Longer run times can impact RNA signal.

Analyte Plex Total Number of FOVs per Run
100 200 400 800 1200 1500
RNA 1000 3.5 4 5 7.5 11* 14* (NR)
Protein 64 1 1.5 2 3 4* 4.5*
Total Tissue Area (mm2) 26 52 104 209 313 392
All times in days. * The current maximum number of FOVs for a single flow cell is 999 (commercial software v1.2). This number is increased to 1149 FOVs in the next software release.
NR: Not recommended

We recommend users to stay within the provided turnaround time guidance.   

Thank you for choosing CosMx. If you have any further questions or need assistance, please don’t hesitate to contact our Support team (Support@nanostring.com).  

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For research use only. Not for use in diagnostic procedures.