GSM8516368: Ileum, with in situ Polyadenylation; Mus musculus; OTHER
Cryosection was obtained from the Ileum of a male, 13 weeks old mouse. The tissue section (10 µm thick) was processed with a modified Stereoseq (STOmics) protocol. The section was mounted onto STOmics mini chip chip ID C02927D611) and fixed in freshly prepared methacarn (60% methanol, 30% glacial acetic acid, 10% chloroform) for 15 minutes at room temperature. Staining and imaging was performed according to the Stereoseq/STOmics protocol. 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 12-minute enzymatic permeabilization step was conducted before continuing with the standard STOmics library preparation to generate cDNA and sequencing libraries. The cDNA ibraries were prepared following the stereoseq/STOmics protocol instructions. sequencing was carried out on a Complete Genomics DNBSEQ-T7 Sequencer using the DNBSEQ-T7 High-throughput Sequencing Set (FCL PE100) and the associated STEROmics primer set. The sequencing run consisted of a 50 bp read 1 (with dark cycles from bases 26 to 40), a 100 bp read 2, and a 10 bp index read.