GSM9463185: Stereoseq heart and testis, with in situ polyadenylation; Mus musculus; OTHER
Frozen testis from DBA/2J mice and frozen reovirus-induced myocarditic hearts collected at 7 days post infection (dpi) following oral gavage with Reovirus type 1 Lang (T1L) were processed for Spatial Total RNA Sequencing using a modified Stereoseq (STOmics) workflow. Tissues were cryosectioned at 10 µm thickness and mounted onto a 1 cm × 1 cm STOmics Stereoseq chip. Sections were fixed in ice-cold methanol for 30 min at −20°C, followed by staining and imaging according to the manufacturer's Stereoseq/STOmics protocol. In situ polyadenylation was then performed using yeast poly(A) polymerase (yPAP; Thermo Scientific): capture areas were equilibrated with 1× wash buffer, incubated with yPAP enzyme mix at 37°C, and the enzyme was removed by washing. A 15-min enzymatic permeabilization step was subsequently performed prior to continuing with the standard STOmics library preparation workflow 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.