GSM6820633: regeneration_6 time-point; Schmidtea mediterranea; OTHER
Samples of different treatments were fixed using methacarn (6 mL methanol, 3 mL chloroform, 1 mL glacial acetic acid). Tissue sections adhered to the Stereo-seq chip surface (Chen et al., 2022), and then incubated at 37 °C for 3 min. After 30 min fixation at -20 °C by methanol (Sigma, 34860), ssDNA of tissues was stained with nucleic acid dye (Thermo fisher, Q10212) and imaged under a fluorescence microscope. 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, and then 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 were stopped by adding 0.02% SDS, followed by amplifying using KAPA HiFi Hotstart Ready Mix. PCR products were purified and used to generate DNBs. Finally, DNBs were sequenced (50 bp for read 1, 100 bp for read 2) on the MGI DNBSEQ-Tx sequencer.