Transcriptomics mIF Solution

Stereo-seq mIF Product Note Stereo-seq Permeabilization Set User Manual Stereo-seq Transcritpomics Set for mIF User Manual

Solution Overview

Combining whole transcriptomics, protein detection, and tissue morphological information on the same tissue section allows for in-depth spatial multi-omics discovery. Stereo-seq v1.3 mIF solution enables spatial visualization of multiple proteins in single-cell resolution on top of the unbiased whole transcriptome information on the same tissue section. Without affecting mRNA capturing, the additional detected protein information can be integrated with gene expression data for in-depth evaluation of valuable samples, and to parse complex pathological and physiological processes.

Product Highlight

Offer flexibility in choosing antibodies based on your research needs and project design

Deliver single-cell resolution results across different species enabling more robust and translatable scientific discoveries

Detect up to 4 protein biomarkers, provide unbiased high-resolution whole transcriptomics information and assess RNA and protein spatial distributions in cells on the same fresh frozen tissue

Improved workflow and upgraded bioinformatic pipeline providing a user-friendly experience

Demo Data

Mouse testis

Tissue area: 17.06 mm²
Total reads: 406,753,020
Valid CID Read: 314,138,027
Median gene type count: 541 (bin20)
Cell count:198,248
Total transcriptome: 211,038,433
Median MID count per cell: 410
Median gene type count per cell: 349
Chip size: 1 cm x 1 cm

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Mouse brain

Tissue area: 27.95 mm²
Total reads: 1.75 G
Valid CID Read: 1,267,486,138
Median gene type count: 282
Cell count:70,035
Total transcriptome: 708.29 M
Median MID count per cell: 1,133
Median gene type count per cell: 732
Chip size: 1 cm x 1 cm

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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 conduct multiplex immunofluorescence(mIF) staining using customized or pre-designed antibody panel 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 products obtained from the Stereo-seq Transcriptomics for mIF application workflow.

Stereo-seq Transcriptomics FF 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.

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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.

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

Catalog Number	Catalog Number (US use only)	Set Name	Specifications	Version	Description
211SP11118	211SP11118-CG	Stereo-seq Permeabilization Set for Chip-on-a-slide V1.1	8 RXN	V1.1	For determining permeabilization parameters to optimize mRNA capture.
211ST13114	211ST13114-CG	Stereo-seq Transcriptomics Set for Chip-on-a-slide V1.3	4 RXN	V1.3	For generating a spatially-resolved 3' mRNA library from biological tissue sections
211ST13004	211ST13004-CG	Stereo-seq Transcriptomics Set for Chip-on-a-slide (0.5cm*0.5cm) V1.3	4 RXN	V1.3
111KL11160	111KL11160-CG	Stereo-seq 16 Barcode Library Preparation Kit	16 RXN	V1.1	Designed for library preparation of samples using Stereo-seq technology, enables the addition of sample barcodes and library construction.
301AUX001	301AUX001-CG	Stereo-seq PCR Adaptor	2 EA	/	Compatible with PCR thermal cycler as a heating unit.

FAQ

Q Can different antibodies be added in the mIF staining process? Which sources of antibodies have been tested so far?

A

The primary antibody should be mixed with different primary antibodies of different origins (primary antibody and secondary antibody should be of the same origin), and so far only primary antibodies of mouse/rabbit/rat origin have been tested.

Q When performing antibody titrations on microscopic glass slides, how many tissue sections can be mounted on one slide?

A

It is recommended to mount only one tissue section per microscopic glass slide to prevent the tissue from drying out during subsequent operations.

Q Is Stereo-seq mIF workflow applicable on any chip sizes other than 1cm x 1cm?

A

Currently not, but we are in the process of testing Stereo-seq mIF workflow on other chip sizes besides 1cm x 1cm.

Q Are there any recommended blocking solutions for non-human species?

A

We recommend to use serum and FCR blocking reagent for preparing the blocking solution. For non-human/mouse species, we recommend to substitute FcR blocking reagent with nuclease-free water.

Q Have other secondary antibodies been tested besides Alexa Fluro Plus 2nd antibodies?

A

We have only tested Alexa Fluro Plus 2nd antibodies.