Jan 26, 2023
Spatial Transcriptomics for FFPE utilizing 10x Genomics Visium
- Andrew Houston1,
- Siqi Chen1,
- Feng Chen1
- 1Department of Medicine, Washington University in St Louis
Protocol Citation: Andrew Houston, Siqi Chen, Feng Chen 2023. Spatial Transcriptomics for FFPE utilizing 10x Genomics Visium . protocols.io https://dx.doi.org/10.17504/protocols.io.kxygx95ezg8j/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: January 18, 2023
Last Modified: January 26, 2023
Protocol Integer ID: 75508
Abstract
To detect gene expression spatially mapped onto formalin-fixed paraffin-embedded tissue samples adapted from the 10x Genomics Visium FFPE v1 protocol.
Quality Assessment
Quality Assessment
Follow the tissue preparation guide as outlined below to determine if your starting FFPE tissue block is acceptable for the 10x Visium platform
Start here
Utilize Visium Tissue Section Test Slide (PN-2000460) to do a quality assessment of each sample
Be sure to trim a few sections and place in a 1.5ml tube for RNA quality check (no more than 40μm)
Follow the steps starting on pg. 10 of the above user guide
Perform all deparaffinization/staining/imaging steps utilizing the test slide
Follow the protocol as described by 10x here
Perform RNA extraction using the Qiagen RNeasy FFPE kit (Cat no. 73504)
Follow the steps outlined in the RNeasy Handbook which can be found below
HB-0375-006_HB_RNeasy_FFPE_0721_WW (1).pdf Start with your pre-cut sections in a 1.5ml tube on pg. 15
After RNA extraction test the concentration of your samples using a nanodrop
Determine the DV200 value of your samples utilizing the Agilent 4200 TapeStation system or similar quantitative assessment
If utilizing Agilent TapeStation to prepare your RNA samples utilize this guide
For first time analysis of DV200 you can set up your parameters as described here
Visium FFPE v1
Visium FFPE v1
If the tissue adheres to the slide and has a DV200 value >50% that sample can be used for the Visium Spatial Transcriptomics platform
Utilize the CG000408 linked above to section onto the Visium Spatial Gene Expression Slide (PN-2000233)
After sectioning start the protocol utilizing CG000409 linked above to perform deparaffinization/staining/imaging steps
Be sure to either store your slide or perform decrosslinking to proceed with the rest of the protocol outlined in the next step
If decrosslinking is complete, utilize the following user guide outlined here
Start probe hybridization on pg. 39
Follow the sequencing requirements on pg. 60 to generate your Visium libraries