GSM8032726: AAV-Clu,mouse heart,stero-seq; Mus musculus; OTHER
10-micron sections were taken from the largest cross-section, attached to the Stero-seq chip, and then incubated at 37°C for 3 min. Followed by 30 min fixation in methanol at -20 °C (Sigma, 34860), the ssDNA of tissues was stained with nucleic acid dye (Thermo, Q10212) and imaged. Tissue sections were permeabilized using 0.1% pepsin (Sigma, P7000) in 0.01M HCl buffer and incubated at 37°C for 15 min. DNA nanoballs (DNBs) on the chip captured released RNA from tissues, then were reverse transcribed at 42°C overnight. After reverse transcription, tissues were digested with tissue removal buffer (10 mM Tris-HCl, 25 mM EDTA, 100 mM NaCl, 0.5% SDS) at 37°C for 30 min cDNA-containing chips were subjected to Exonuclease I (NEB, M0293L) at 37°C for 1h. PCR reactions with KAPA HiFi Hotstart Ready Mix (Roche, KK2602) were performed to amplify cDNAs. Qubit™ dsDNA Assay Kit (Thermo, Q32854) was used to quantify the concentrations of PCR products. cDNAs were fragmented with in-house Tn5 transposase at 55°C for 10 min, which was stopped by adding 0.02% SDS, then amplified using KAPA HiFi Hotstart Ready Mix. PCR products were purified and used to generate DNBs.