Spatial transcriptomics (10x Visium) of human NSCLC lesions and non-involved tissue
Spatial transcriptomics (10x Visium) of human NSCLC lesions and non-involved tissue
Tissues were frozen in dry-ice cooled isopentane and stored in air-tight tissue cryovials at -80°C. Prior to undertaking any spatial transcriptomics protocol, the tissues were embedded in an optimal cutting temperature compound (OCT) and tested for RNA quality with RNA integrity number (RIN). Tissues with RIN values >7 were cryosectioned in a pre-cooled cryostat at 10 μm thickness. Two consecutive sections were cryosectioned at 10 μm thickness in a pre-cooled cryostat and transferred to the four 6.5mm x 6.5mm capture areas of the gene expression slide. Slides were fixed in methanol for 30 minutes prior to staining with H&E and then imaged using the Nanozoomer slide scanner. The tissues underwent permeabilization for 24 minutes.  Reverse transcription and second strand synthesis was performed on the slide with cDNA quantification using qRT-PCR using KAPA SYBR FAST-qPCR kit (KAPA Biosystems) and analysed on the QuantStudio (ThermoFisher). Following library construction, these were quantified and pooled at 2.25 nM concentration. Reverse transcription and second strand synthesis was performed on the slide with cDNA quantification using qRT-PCR using KAPA SYBR FAST-qPCR kit (KAPA Biosystems) and analysed on the QuantStudio (ThermoFisher). Following library construction, these were quantified and pooled at 2.25 nM concentration.