GSM8153571: Sen.Res.ICI_spatial; Mus musculus; OTHER
The frozen tissue was embedded in an Optimal Cutting Temperature compound (OCT, Sakura Tissue-TEK) on dry ice and stored at -80 °C. Before proceeding with the complete protocol, the Tissue Optimization Kit was employed according to the manufacturer's instructions. The fluorescent footprint was then imaged using a Metafer Slide Scanning Platform (Pannoramic MIDI) to determine the optimal permeabilization time. Sections of the OCT blocks, cut at a thickness of 10 μm using a pre-cooled cryostat, were transferred to fit the oligo-barcoded capture areas (6.8 x 6.8 mm²) on the BMKMANU S1000 Gene Expression Slide. The Gene Expression Slide, along with the tissue, was fixed and stained with hematoxylin and eosin (H&E) and imaged at 40X magnification using a Pannoramic MIDI microscope. Tile scans of the entire array were acquired and the images were merged. The sequence libraries were prepared in accordance with the manufacturer's instructions (BMKMANU, Library Construction Kit, ST03002-34) using TruSeq Illumina libraries. The libraries were sequenced on a NovaSeq (Illumina) by Integragen (Evry) with a minimum sequencing depth of 150,000 read-pairs per spatial spot. Sequencing was carried out following the recommended protocol: read 1 (28 cycles), i7 index read (10 cycles), i5 index read (10 cycles), and read 2 (50 cycles), resulting in a total of 300 million to 400 million sequenced reads. BMKMANU, Library Construction Kit, ST03002-34