DNeasy PowerSoil Pro Kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Do not use bleach to clean the inside of the PowerVac™ Manifold or to rinse the PowerVac™ Mini Spin Filter Adapters when attached to the manifold.

Upstream tips
Do not use bleach to clean the inside of the PowerVac™ Manifold or to rinse the PowerVac™ Mini Spin Filter Adapters when attached to the manifold.

Publication protocol

Upon arrival, wild-harvested animals were allowed to acclimate in 0.22 μm-filtered artificial seawater (ASW) for 3–4 h. All steps to isolate fungi were carried out under a laminar hood, in aseptic conditions, using sterile materials. Briefly, after sterilization of the Ciona tunic with 70% ethanol and washing with sterile ASW (41), gut was excised surgically (aseptically) from five animals and disrupted using a Dounce homogenizer to liberate bacteria and fungi from the mucosal surface of the organ. Host tissue was separated by processing through a 40 μm filter at 1200 × g for 10 min. Gut microorganisms were collected by pelleting at 500 × g or 10 min, washing and re-suspending in 1 ml ASW. An aliquot of this suspension (300 μl) was plated onto Yeast Peptone Dextrose (YPD) Agar plates (2% BactoPeptone, 1% Yeast extract, 2% dextrose, and 2% Agar) dissolved in ASW with Penicillin/Streptomycin (200 U/ml and 0.2 mg/ml, respectively; Fisher BP295950) antibiotics. Fungi were grown for 7–10 days before clonal growth was established and maintained by replica-streaking on YPD plates without antibiotics. Each fungal isolate was then inoculated in 5 ml YPD liquid medium, grown for 1 week in an orbital shaker at 20°C and then mixed with glycerol (10% final concentration) for long-term storage at −80°C.

Fungal species were identified by sequencing (Sanger) across the interspersed spacer (ITS) region of the 18s rRNA gene. Briefly, using Dneasy PowerSoil kit (Qiagen, Cat#12888), DNA extraction was performed on fungi grown in liquid medium for 5–7 days; fungal-specific18s rRNA amplicons were generated with standard ITS primers [ITS1 and ITS4, (42)]. Sequence data were deposited in GenBank and accession numbers are reported in Supplementary Table 1.

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