Stereo-CITE Proteo-Transcriptomics solution

Stereo-CITE Proteo-Transcriptomics Set User Manual Stereo-seq Permeabilization User Manual For Stereo-CITE Stereo-CITE product note

Solution Overview

Combined with Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-seq), STOmics Stereo-CITE Proteo-Transcriptomics Set is intended for simultaneous detection of the whole transcriptome and 100+ plex protein on the same tissue section.
Stereo-CITE facilitates the study of tissue heterogeneity and associated biological significance in complex (disease) tissues, leading to a broader understanding of spatial omics studies.

Product Highlight

No auto-fluorescence interference or antigen instability caused by multiple rounds of detection

High detection efficiency of 100+ plex proteins with antibody combinations of your choice from in-house validated vendor

True single-cell resolution for both transcriptome and protein analysis

Unbiased spatial profiling of whole transcriptome and hundreds of protein markers on the same tissue section

Workflow

Sample Preparation

Stain and image

mRNA capture & cDNA synthesis

Library construction

Sequencing

Data analysis & visualization

Sample Preparation

Embed, section, and mount fresh frozen tissue sections on the Stereo-Chip Slide.

Stereo-seq Sample Preparation Guide for Fresh Frozen Samples on Stereo-seq Chip Slides

Stain and image

Carry out tissue fixation and DAPI staining (nuclei staining, fluorescence) to visualize the tissue under epi-fluorescence microscopes. An upright microscope is recommended for optimal image generation.

mRNA capture & cDNA synthesis

To extract mRNA from fresh frozen tissue, the tissue undergoes permeabilization. This process enables mRNA to bind to DNB-patterned array chips (Stereo-seq Chip T) that are preloaded with spatially-barcoded probes designed to capture and prime poly-adenylated mRNA from the tissue. Each cDNA synthesized at a specific spot (500nm resolution) is attached to its spatially barcoded probe, enabling the subsequent mapping of gene expression within a tissue section after sequencing.

Library construction

Construct a whole-transcriptome library by amplifying and purifying the cDNA and ADT (antibody-derived tags) products obtained from the Stereo-CITE workflow.

Stereo-CITE V1.1 Proteo-Transcriptomics Library Preparation User Manual

Sequencing

Stereo-seq libraries can be sequenced on either DNBSEQ-T7RS or DNBSEQ-G400RS platforms using PE75 Stereo-seq Visualization Reagent set.

Expand All Retract

Data analysis & visualization

Run your sequencing data on the Stereo-seq Analysis Workflow (SAW), which involves Linux command line-based pipelines, to map the sequenced reads back to their spatial locations on the tissue section, and then interactively display the results on StereoMap visualization software, a Windows/MacOS compatible desktop application.

Demo Results

Exploring with a broader perspective

Facilitating multi-omics joint analysis

Mouse Spleen Protein Clustering Result (Cellbin)
Capture area size: 1 cm x 1cm

Mouse Spleen Single-cell Clustering Result (Cellbin)
Capture area size: 1 cm x 1cm

Mouse Spleen Protein Expression Heatmap
Capture area size: 1 cm x 1cm

Mouse Spleen Gene Expression Heatmap
Capture area size: 1 cm x 1cm

Mouse Spleen DAPI Staining Image
Capture area size: 1 cm x 1 cm

Mouse Thymus Gland Spatial Protein Expression Profile
Capture area size: 1 cm x 1cm

Mouse Thymus Gland Gene Expression Profile
Capture area size: 1 cm x 1cm

Mouse Thymus Gland DAPI Image
Capture area size: 1 cm x 1cm

Mouse Thymus Gland Transcriptom Clustering Result (Cellbin)
Capture area size: 1 cm x 1cm

Mouse Thymus Gland Protein Clustering Result (Cellbin)
Capture area size: 1 cm x 1cm

Perform Stereo-CITE in your own laboratory with Stereo-seq Kit Solutions

Stereo-seq Permeabilization Set for Chip-on-a-slide V1.1
Catalog Number	Part Name	Part Number	Specification	Description
211SP11118	Stereo-seq Permeabilization kit	211KP11118	8 RXN	For determining permeabilization parameters to optimize mRNA capture
	STOmics Accessory Kit	1000033700	5 PCs
	Stereo-seq Chip P Slide (1cm*1cm)	210CP118	8 EA

Stereo-CITE Proteo-Transcriptomics Set V1.1
Catalog Number	Part Name	Part Number	Specification	Description
211PT11114	Stereo-seq Transcriptomics T kit	211KT13114	4 RXN	For generating a spatially-resolved 3' mRNA library and ADT (protein) library from the same biological tissue sections
	STOmics Accessory Kit	1000033700	5 PCs
	Stereo-seq Chip T Slide (1cm*1cm)	210CT13114	4 EA
	Stereo-seq Protein Assisted Kit	212KA11114	4 RXN

FAQ

Q Can pre-mixed commercially available antibody cocktails be used for this experiment?

A

Yes, currently validated mouse antibody cocktails include the TotalSeq™-A Mouse Universal Cocktail, V1.0 (Biolegend, Cat. No. 199901, for mouse samples, consisting of 119 antibodies) and the TotalSeq™-A Human Universal Cocktail, V1.0 (Biolegend, Cat. No. 399907, for human samples, consisting of 154 antibodies).

Q The current protocol uses PFA to fix the sections at room temperature after tissue sectioning. Can the sample be fixed with PFA before OCT embedding for subsequent experiments?

A

Currently, the Stereo-CITE Proteo-Transcriptome protocol is not compatible with PFA-fixed samples and is only suitable for freshly embedded samples.

Q How many proteins can the current product detect simultaneously? What are the main research areas of these protein targets?

A

This product is compatible with three types of protein antibody panels for detecting different numbers of proteins.

(1) Commercial protein panels, such as TotalSeq™-A Mouse Universal Cocktail, V1.0 (Biolegend, Cat. No. 199901) and TotalSeq™-A Human Universal Cocktail, V1.0 (Biolegend, Cat. No. 399907). These panels can detect 119 and 154 proteins (excluding isotype controls), respectively. The proteins targeted by these panels are mainly cell surface proteins, including those used for immune cell typing.

(2) Freely mixed TotalSeq ™ -A series antibodies, with the number of proteins depending on your selection;

(3) Freely mixed self-conjugated antibodies. For example, you can use the Stereo-seq self-conjugation kit to conjugate your primary antibodies of interest with antibody-derived-tag (ADT) oligonucleotides. After verifying that these antibodies are compatible with the Stereo-CITE workflow, you can freely mix them to achieve the desired number of proteins.