Tissue homeostasis at the root of Metazoa: transcriptional control of whole-body regeneration in sponges
Description
Current data supports the results of study "Tissue integrity at the root of Metazoa - transcriptional landscape of whole-body regeneration in sponges" (2025) by Melnikov Nikolai, Kseniia V. Skorentseva, Alexander V. Ereskovsky, Ilya E. Borisenko, Fyodor V. Bolshakov, Andrey I. Lavrov. Folder "R" consists of R scripts and data needed to reproduce DE and coexpression analysis of RNAseq experiment regarding reaggregation of cells in demosponge Halisarca dujardinii. Folder "Primmorphs" contains original photos of histological semithin sections of aggregates.
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Steps to reproduce
All information related to sampling, processing and data manipulation are provided in the manuscript and its additional files. Histological studies were used to describe changes in cell composition and tissue structure occurring during reaggregation. For this purpose, samples were fixed for 2 hours at 4°C with the mixture of 2.5% glutaraldehyde (Electron Microscopy Science, 16020) and 0.1% of ruthenium red (Sigma-Aldrich) and post-fixed for 1 hour at room temperature with the mixture of 1% OsO4 (Electron Microscopy Science, 19100) and 0.1% of ruthenium red (Sigma-Aldrich). Both fixation and post-fixation were performed in modified 0.1M Na-Cacodylate buffer (0.1M Na-Cacodylate, 85.55 mM NaCl, 5 mM CaCl2, 5 mM MgCl2, pH 7.0-7.5 pH)37. Post-fixed specimens were dehydrated and embedded in Epon/Araldite epoxy embedding media (Electron Microscopy Science, 13940). Semithin sections (1 µm) were made using LKB V and Leica UC6 ultramicrotomes and stained with 1% toluidine blue – 0.2% methylene blue mixture for 1-1.5 min at 60°C. Semithin sections were studied under a Leica DMBL microscope equipped with the Evolution LC photo capture system. For RNA sequencing, culture samples were centrifuged and lysed in 1 ml of ExtractRNA (Evrogen, BC032). During the lysis, specimens were placed at room temperature for 15-20 min and homogenized with sterile pestles; then, they were cooled at +4°C for 20 min, placed at -20°C for at least 2 h and finally stored at -80°C. RNA extraction was performed using ExtractRNA (Evrogen, BC032) followed by purification with CleanRNA Standard kit (Evrogen, BC033). Resulting samples were checked for RNA integrity by electrophoresis in 1% agarose gel in Tris-acetate-EDTA buffer (Evrogen, PB022). Purified RNA samples in RNAse-free water were freezed and stored at -80°C until library preparation. Polyadenylated fraction of total RNA was purified with NEBNext Poly(A) mRNA Magnetic Isolation Module (New England Biolabs). Libraries were constructed with NEBNext Ultra II RNA Library Prep Kit and sequenced on the Illumina NovaSeq6000 at paired-end mode with 150 bp read length. Sequencing results were analyzed using multiQC v1.17. Reference transcriptome was used to estimate transcript counts using kallisto v0.50.147 with standard options for paired end reads. Downstream analysis was performed using DeSeq2, Mfuzz and clusterprofileR R packages.