Sanger Tree of Life Sample Homogenisation: Sponge Squeezing v1

This protocol is for the homogenisation of sponge samples for HMW DNA extraction and/or HiC, intended for long read sequencing. In the phylum Porifera, most marine demosponges (Demospongia) species possess a siliceous skeleton and spicules which can compose a high volume of the total biomass. Silica deposition is a fundamental process in sponges allowing sponges to form complex three-dimensional structures connected by spongin. However, these hard siliceous spicules can hinder the mechanical lysis of tissue and restrict the access of reagents like lysis buffer and Proteinase K to the cells, which are needed for DNA release and purification. Standard powermashing of these tissue types is often not sufficient to extract a sufficient yield of HMW gDNA. A “squeeze” method is described here to enrich for intact cells (eukaryotic and prokaryotic) prior to DNA extraction from marine sponge tissues. This protocol was adapted from that designed by Jose Lopez. This process is highly effective for the disruption of flash frozen sponges 150-200 mg in mass from Demospongia covered by the Tree of Life Programme. This protocol can also be used on glass sponges Hexactinellida, but alterations should be made depending on morphology. The output of this protocol is a sponge cell pellet that can be directed towards the Sanger Tree of Life HMW DNA extraction: Manual MagAttract or Manual Plant MagAttract protocols.