Spatial Transcriptomics for FFPE utilizing 10x Genomics Visium

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

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