title: GSM8507231: Large Intestine, with in situ Polyadenylation; Mus musculus; OTHER
library_construction_protocol: Cryosections were obtained from four distinct locations of the intestine of the same individual (male, 13 weeks old) — the proximal small intestine, ileum, cecum, and large intestine. Tissue sections (10 µm thick) were processed using either the standard Visium protocol or a modified protocol. For the modified protocol, sections were mounted onto Visium Spatial Gene Expression v1 slides and fixed in freshly prepared methacarn (60% methanol, 30% glacial acetic acid, 10% chloroform) for 15 minutes at room temperature. H&E staining was performed following the Visium protocol, and the slides were transferred into the Visium Slide Cassette.  In situ polyadenylation was performed using yeast Poly(A) Polymerase (yPAP; Thermo Scientific). Capture areas were equilibrated with 1X wash buffer, followed by the addition of yPAP enzyme mix. The chambers were sealed and incubated at 37°C for 25 minutes. After enzyme removal, a 30-minute enzymatic permeabilization step was conducted before continuing with the standard Visium library preparation to generate cDNA and sequencing libraries. For the standard Visium protocol, H&E staining and imaging were directly followed by permeabilization. The cDNA ibraries were prepared following the 10x Visium protocol instructions. Sequencing was performed on a NextSeq 2K platform with a P3 200bp kit, with reads allocated as follows: 28 bp for read 1, 10 bp for index 1, 10 bp for index 2, and 190 bp for read 2.