Dual RNA Isolation from Host-Fungus Samples

We designed an RNA isolation protocol for isolation of both fungal and murine RNA from the same sample based on step-wise isolation of host and fungal RNA (Suppl. Fig. 5). First, organs were aseptically homogenized in RLT buffer (Qiagen) with 1% β-Mercaptoethanol (β-ME) on ice water using an Ika T10 basic UltraTurrax homogenizer. Then, homogenates were centrifuged at 3,000 g at 4 °C for 3 min. The supernatant was used for mouse tissue RNA isolation with the RNeasy Mini Kit (Qiagen) as described by the manufacturer. The remaining pellets were vortexed on a mini-beadbeater (Precellys) for 5 sec at 5000 m/s in 1 ml RLT buffer (Qiagen) with 1% β-ME. After centrifugation at 4 °C, fungal RNA was isolated from the remaining pellets as previously described24 (link). The RNA quality was determined using a Bioanalyzer (Agilent Inc.), the quantity was measured with a Nanodrop ND1000 (Peqlab).