title: Illumina NovaSeq 6000 sequencing; The human gastrointestinal tract through space and time- Fetal Spatial transcriptomics
design: The human gastrointestinal tract through space and time- Fetal Spatial transcriptomics
library_construction_protocol: Human fetal gut samples were obtained from the Human Developmental Biology Resource (HDBR, www.hdbr.org). The maternal consent was obtained through Newcastle hospital (the consent REC reference 18/NE/0290- IRAS project ID: 250012). Fetal gut tissue was embedded in optimal cutting temperature medium (OCT) and frozen on an isopentane-dry ice slurry at -60°C, and then cryosectioned onto SuperFrost Plus slides at a thickness of 10 μm.  10x Genomics Visium protocol was applied on OCT embedded fresh frozen samples. All tissues were sectioned using the Leica CX3050S cryostat and were cut at 10um. The samples were selected based on morphology, orientation (based on H&E) and RNA Integrity Number (RIN) that was obtained using Agilent2100 Bioanalyzer. Tissue Optimisation was performed to obtain permeabilisation time for foetal tissue (12 min) and after optimisation the Visium spatial gene expression protocol from 10X Genomics was performed using Library Preparation slide and following the manufacturer's protocol.  After transcript capture, Visium Library Preparation Protocol from 10x Genomics was performed. All images for this process were scanned x40 on Hamamatsu NanoZoomer S60.