A 4 81

1.0 mL of either N. californiae and A. robustus was inoculated by sterile syringe into 0.1 g of reed canary grass substrate and 10 mL of complex media (Davies et al., 1993 (link)) (“MC”) in each Hungate tube with 100% CO₂ headspace and grown anaerobically at 39°C for 48 h. After the growth period, a total of 24 replicates of each species were subjected to a 48°C water bath for 15 min. The fungi were harvested in replicates of four at 15 min intervals starting immediately before heat shock (control group) up to 60 min after the completion of the heat shock. Standard good practices for working with RNA were followed during all steps. The cultures were transferred from the Hungate tubes to 15 mL Falcon tubes at the time of harvest. They were then centrifuged for 7 min at 4°C and 3,220 g in a swinging bucket rotor (Eppendorf™ A-4-81). One milliliter of RNAlater® (Sigma-Aldrich®) was added to each of the pellets using sterile, filter pipette tips. The samples were then vortexed for 5 s to thoroughly mix the pellet and stabilization solution. The Falcon tubes containing the pellets with RNAlater®, were stored at −20°C until extraction.

Samples were thawed on ice and then centrifuged at 3,220 × g at 4°C using a swinging-bucket rotor (Eppendorf A-4-81) for 10 min. RNAlater was decanted. The pellets were transferred into 2-ml screw-cap tubes containing 1.0 ml of autoclaved 0.5-mm zirconia/silica beads (Biospec) and 600 μl buffer RLT (Qiagen, Hilden, Germany) with 1 vol% 2-mercaptoethanol (Sigma-Aldrich, St. Louis, MO). The tube was briefly vortexed, and then the cells were lysed using the Biospec Mini-Beadbeater-16 for 45 s. The tubes were placed on ice for 30 s. Following lysis, the tubes were centrifuged for 3 min at 13,000 × g and 20°C using a microcentrifuge (Eppendorf 5424). The supernatant was removed using gel-loading tips (Fisher Scientific, Waltham, MA) to maximize yields and centrifuged again to remove residual cell debris for 2 min at 20,000 × g. The supernatant from each tube was then transferred into 2-ml round-bottom sample tubes (Qiagen catalog number 990381). RNA was extracted using a QIAcube by following the RNeasy Mini protocol for animal cells with QIAshredder homogenization and optional on-column DNase digest.
Quantity and quality of RNA was assessed by a QuBit fluorometer and TapeStation (Agilent), respectively. All RNA integrity number equivalents (RIN^e) were above 6.0, assessed by either eukaryotic or prokaryotic ribosomal markers for cocultures.