GSM8517210: Ileum, with in situ Polyadenylation, Stereoseq (STOmics); Mus musculus; OTHER
Cryosection was obtained from the Ileum of a female, 17 weeks old mouse. The tissue section (10 µm thick) was processed with a modified Stereoseq (STOmics) protocol. The collected section had visible signs of neoplasia adjacent to the lumen. The section was mounted onto STOmics mini chip chip ID C02929E514) 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.