GSM6046661: P3-TM-ST; Homo sapiens; OTHER
The resected tissues were trimmed to cubes with diameter of 0.6-1.0 cm. Use sterile filter paper to suck out blood and liquid. The tissue was completely immersed in OCT and placed in liquid nitrogen for cryoembedding and stored in – 80℃.  Then, prepare 10 frozen sections (10 um) for every sample and extract RNA with Qiagen kit for 2100 detection. Samples with RIN>7 were eligible. The Visium Spatial Tissue Optimization Slide & Reagent kit (10X Genomics, US) was used to optimize permeabilization conditions for the tissue according to Visium Spatial Tissue Optimization User Guide (CG000238, https://support.10xgenomics.com/). Briefly, the Visium Spatial Tissue Optimization workflow includes placing tissue sections on 7 Capture Areas on a Visium Tissue Optimization slide. The sections are fixed, stained, and then permeabilized for different times. mRNA released during permeabilization binds to oligonucleotides on the Capture Areas. Fluorescent cDNA is synthesized on the slide and imaged. The permeabilization time that results in maximum fluorescence signal with the lowest signal diffusion is optimal. If the signal is the same at two time points, the longer permeabilization time is considered optimal. Visium Spatial Gene Expression Reagent Kits User Guide (CG000239) was used to generate Visium Spatial Gene Expression libraries. 4 capture areas, each with ~5000 unique gene expression spots, were included. Each capture area is 6.5 x 6.5 mm and is surrounded by a fiducial frame for a total area of 8 x 8 mm. A readable label with a serial number defines the active surface of the slide. Tissue sections are always placed on the capture areas on the active surface.Sequencing was performed with a Novaseq 6000 PE150 platform according to the manufacturer's instructions (Illumina, US) at an average depth of 300 million read-pairs per sample. Enzymatic fragmentation and size selection are used to optimize the cDNA amplicon size. P5, P7, i7 and i5 sample indexes, and TruSeq Read  2 (read 2 primer sequence) are added via End Repair, A-tailing, Adaptor Ligation, and PCR. The final libraries contain the P5 and P7 primers used in Illumina amplification. A Visium Spatial Gene Expression library comprises standard Illumina paired-end constructs which begin and end with P5 and P7. The 16 bp Spatial Barcode and 12 bp UMI are encoded in Read 1, while Read 2 is used to sequence the cDNA fragment. i7 and i5 sample index sequences are incorporated. TruSeq Read 1 and TruSeq Read 2 are standard Illumina sequencing primer sites used in paired-end sequencing.