Visium Spatially Resolved Transcriptomics of Glioblastoma Samples

Description

Methods

Methods: 

Spatially resolved Transcriptomics: Quality Assessment Tissue

Considering that FFPE blocks typically contain tissue exceeding the Visium Gene Expression Slides' capture area of 6.5 x 6.5 mm, the tissue's area of interest was determined by slicing sections of 5 μm thickness with a microtome, floating these sections in a 42 °C water bath, and transferring them onto glass slides. Following H&E staining, the tissue was examined under the EVOS microscope, and the interest area was selected. 

Spatially resolved Transcriptomics: Tissue Preprocessing

The initial stage of the Spatial Transcriptomics methodology utilized the 'Visium Spatial Gene Expression for FFPE – Tissue Preparation Guide' (CG000408 | Rev A). Any modifications to steps are detailed explicitly in the subsequent descriptions. Hydration and Trimming: Without performing a tissue adhesion test due to no apparent tissue detachment on glass slides, the FFPE tissue blocks were hydrated in an ice water bath for 20 minutes facing down. Thereafter, using the Thermo Fisher Scientific™ HM355 S automatic microtome set to 10 μm, the blocks were trimmed and cut into 4 μm thick sections. A standard glass slide harvested the section from a 21 °C water catch basin. Excess paraffin and tissue parts were trimmed on the slide with a blade, discarding the leftovers with a thin paint brush. The section was floated in a 42 °C water bath for extension and smoothing of minor tissue wrinkles. After a brief floating period (5-10 seconds), the section was fit onto one of the four capture areas of the Visium slide (PN2000233) by immersing the slide in water and guiding it with a paint brush. The Visium slide, fitted with four tissue sections, was placed on a thermocycler adaptor (PN3000380) and dried using the Eppendorf Mastercycler® X50 thermocycler at 42 °C for three hours. Finally, the slide was stored in a desiccator at room temperature overnight.

Deparaffinization, H&E Staining, Imaging & Decrosslinking:

After heating the Visium slides at 60 °C for two hours, they underwent two 15-minute immersions in xylene, followed by serial dilutions in 100%, 96%, 85%, and 70% ethanol for three minutes each. The slides were finally rinsed in Milli-Q water for 20 seconds. The slides were stained with 1 ml of hematoxylin for three minutes, washed in two successive Milli-Q water baths, treated with 1 ml of bluing buffer for one minute, washed again, and then stained with 1 ml of alcoholic eosin for one minute, followed by another wash. Imaging was carried out with an EVOS M7000 microscope from Thermo Fisher Scientific at 20x magnification in the brightfield setting, utilizing auto-focus for the first image of each capture area. Following imaging, the slide was placed into a Visium slide cassette (PN2000282) with an alignment tool (PN3000433). Pipetting was performed carefully to prevent disturbing the tissue, ensuring full coverage of the capture area and complete removal of leftover fluids. Each well of the cassette was treated twice with 100 μl of 0.1N HCl, then rinsed with 150 μl of pH 9.0 TE buffer, followed by another TE buffer application and incubation at 70 °C for one hour on a thermal cycler. This initiated the library construction's hybridization stage.

Spatially resolved Transcriptomics: Library

Pre-hybridization mix was applied to each well, incubated for 30 min at 37 °C. This involved an overnight incubation of probe hybridization mix at 50 °C, centrifugation, multiple washes, and application of probe ligation mix for one hour at 37 °C. Post-ligation wash buffer was used, followed by several washes. RNase mix and permeabilization mix were each applied and incubated for 30 minutes and one hour respectively at 37 °C, followed by washing and probe extension mix application. 0.08 M KOH was used to elute the probe. The solution was transferred to an 8-tube-strip, and 1 M pH 7.0 Tris-HCl added. The process involved determining cycle numbers for PCR using a qPCR mix. A StepOnePlus Real-Time PCR System was used. Sample Index PCR was performed, followed by cleanup, adding SPRIselect to each sample, and transferring 25 μl to a new tube strip. A second qPCR was performed with the NEBNext® Library Quant Kit for Illumina®. Libraries were diluted and filled into wells for a PCR plate. A qPCR protocol was followed, and molarities for each library obtained via the NEBioCalculator®. The qPCR was used to ensure library construction success and cDNA presence, acting as a substitute for the Agilent Bioanalyzer High Sensitivity chip.

Spatially resolved Transcriptomics: Sequencing

The libraries were sequenced using the NextSeq™ 500/550 device from Illumina®. Libraries were normalized to the same molarity, then combined. The ‘NextSeq System – Denature and Dilute Libraries Guide’ protocol was used. The required amount of the combined library was denatured with an equivalent amount of 0.2 N NaOH, incubated, and neutralized with 200 mM Tris-HCl pH 7. Prechilled HT1 from the Illumina kit was added to the denatured library based on its concentration. Denatured libraries were diluted to a loading concentration using High Output Kits. To prevent under-clustering, a smaller dilution to 2.4 pM was chosen. PhiX control was also denatured, diluted and mixed with the library. The final mix was incubated at 90 °C for a minute, then put on ice while preparing the sequencer. For sequencing, the NextSeq 500/550 High Output Kit v2.5 (75 cycles) was used, with cycles distributed among Read 1, Index 1 and Index 2, and Read 2.