title: Rack1-mediated ferroptosis affects hindgut development in rats with anorectal malformations: Spatial transcriptome insights
design: Rack1-mediated ferroptosis affects hindgut development in rats with anorectal malformations: Spatial transcriptome insights
library_construction_protocol: Embryos were collected from pregnant Wistar rats at gestational day 14–16 under isoflurane anesthesia, following institutional ethical approval (No. 2020PS357K, Shengjing Hospital, China Medical University). Immediately after cesarean section, embryos were rinsed in ice-cold sterile saline to remove blood and blotted dry with RNase-free gauze. Each intact embryo was embedded in pre-cooled optimal cutting temperature (OCT) compound (SAKURA, Japan) in sagittal orientation and snap-frozen in isopentane chilled with liquid nitrogen for 1 min. Samples were stored at −80 °C until sectioning. Spatially barcoded mRNA molecules were captured in situ on 10x Genomics Visium Spatial Gene Expression Slides. Tissue sections (10 µm thick) were fixed in 3.7% formaldehyde, stained with hematoxylin and eosin, and imaged before permeabilization. Permeabilization was optimized for embryonic tissue using 0.5 U/ml collagenase and 0.2 mg/ml BSA in HBSS at 37 °C for 20 min, followed by digestion in 0.1% pepsin in 0.1 M HCl for 7 min. Released mRNA hybridized to the oligo-dT primers within each spatial capture spot. Reverse transcription generated spatially barcoded cDNA molecules, which were enzymatically released using a Proteinase K/PKD buffer (Qiagen) mix at 56 °C for 1.5 h, then collected for library construction. cDNA libraries were prepared following the 10x Genomics Visium Spatial Gene Expression User Guide and previously published protocols (Salmén et al., 2018; Ståhl et al., 2016). Released cDNA underwent second-strand synthesis, in vitro transcription, adaptor ligation, and second reverse transcription to generate indexed sequencing libraries. The libraries were purified, quantified, and quality-checked before sequencing. All spatial library preparation steps were performed on an automated MBS 8000 system (Jemt et al., 2016) by Biomarker Technologies Corporation (Beijing, China).