Tomo-seq data from ear tissue

We used Tomo-seq to investigate the spatial gene expression pattern during ear regeneration. This method is based on cryosectioning of the desired tissue and performing RNA-seq with a CEL-seq approach on individual sections (Kruse et al. 2016). Both ears of the rodents were punched with a 4 mm punch and the ears were harvested after various timepoints: uninjured, 1 hour, 1 day, 5 days, 10 days and 55 days after ear punch. For the non-regenerating rodents, I took the following timepoints: uninjured, 1 day after punch and 10 days after punch. Each timepoint was replicated three times for robust analysis. After collection, one ear was preserved in 4% PFA for future staining, while the other was embedded in Jung tissue freezing medium (Leica), flash-frozen with dry ice, and stored at -80°C. Subsequently, I cryo-sectioned the embedded ears from proximal to distal and sliced them into 96 sections of 20 μm thickness. Every fifth section was taken for further analysis, covering a total span of 9,6 mm. I extracted RNA from each individual section with TRIzol reagent. The sections were processed individually, barcoded and processed for RNA sequencing as previously described (Junker et al., 2014). An Illumina sequencing library was prepared per ear with the TruSeq small RNA sample prep kit (Illumina) and sequenced paired-end by Illumina NextSeq500. The paired-end reads were aligned to the Acomys cahirinus(AcoCah_v1_BIUU), Mus musculus (GRCm38) or Meriones unguiculatus (MunDraft-v1.0) genome using Spliced Transcripts Alignment to a Reference (STAR) (Dobin et al. 2013). The 5’ mate of each pair of 60 bp was used for mapping, discarding all reads that mapped equally well to multiple loci. The 3’ mate of 26 bp was used for barcode information. To obtain a comparable number of reads per section in one sample, a cut off of either 1000 , 5000 , or 10000 (reads per section) was used, resulting in the retention of ±75% sections for the analysis. For the spatial gene expression analysis, read counts were normalized to the total counts per section. Next, the data was renormalized to the mean of total reads across sections to ensure that count numbers roughly corresponded to the number of mapped reads. Afterwards the data was transformed to Z-score values and averaged for the three biological replicates. For the expression level analysis, the raw read counts from the -5 and +5 sections surrounding the wound edge were summed. The section containing the wound edge was marked during sectioning. A comparable area was taken in the uninjured area. For the original paper and more details, see our resource paper published in Genome Research (van Beijnum et al. 2023). The Tomo-seq data generated in this study are available at the NCBI BioProject database under accession number PRJNA898313.