title: GSM8673673: duodenum tissue and colon tissue, CD or HfiD feed 5w, HFD feed 0w; Mus musculus; OTHER
library_construction_protocol: Tissue section preparation, library construction and sequencing for stereo-seq: The procedures were conducted as previously described (Chen et al., 2022). Briefly, 10-μm thick sections of small and large intestine were placed onto a precooled stereo-seq chip for incubation at 37°C for 5 min, followed by fixation in methanol at -20°C for 40 min. The chip with tissue was stained with the Qubit ssDNA labelling and detection kit (Invitrogen, Q10212) for 5 min and subsequently washed with 0.1x SSC buffer (Thermo Fisher, AM9770) supplemented with 0.05 U/μl RNase inhibitor (Solarbio, R8061), before being imaged with an Eclipse Ti-7 microscope (Nikon). RNA extraction from the tissue to the chip was achieved by incubation with 0.1% pepsin dissolved in 0.01M HCl with a pH value of 2 at 37°C for 12 min. The released RNA was reverse transcribed overnight at 42°C using the SuperScript II kit (Thermo Fisher, 18064-014). The cDNA-containing chip was further processed with a tissue removal buffer containing 10 mM Tris-HCl, 25 mM EDTA, 100 mM NaCl, 0.5% SDS and Exonuclease I (NEB, M0293L), before being amplified with the KAPA HiFi Hotstart Ready Mix kit (Roche, KK2602). 20 ng DNA was fragmented with Tn5 transposase and amplified with the KAPA HiFi Hotstart Ready Mix kit. The PCR product was purified with the AMPure XP Beads (Beckman Coulter) and sequenced with an MGI DNBSEQ-Tx sequencer (MGI).