On column dnase digestion

Frozen microbial cell balls were thawed on ice and suspended in ice-cold RLT buffer (Qiagen Ltd., Manchester, UK) containing 2-mercaptoethanol and transferred to a sterile screw cap microfuge tube containing acid-washed Biospec glass beads (0.6 ml). The suspension was mixed with the glass beads and the fungal and bacterial cells were disrupted by alternating shaking (30 s) using a Fast-prep 25 bead beater (MP Biomedicals, Santa Ana, CA) and incubating 1 min on ice (repeated 3 times). The beads were allowed to settle and the supernatant was transferred to a sterile microfuge tube. The disrupted cells were centrifuged (13000 × g, 2 min) and the supernatant transferred to a sterile microfuge tube. An equal volume of 70% ethanol was added and the RNA was extracted and purified using an RNeasy Mini Kit (Qiagen) with the use of an on-column DNAse digestion (Qiagen). The quality of the RNA was checked by formaldehyde agarose-gel electrophoresis. The RNA concentration of each sample was measured spectrophotometrically (Nanodrop 1000, Thermo Scientific, Fisher Scientific UK Ltd, Loughborough, Leics., UK) and stored at −20°C.

RNA was extracted from cells using the RNeasy mini kit (Qiagen, Germantown, MD) and DNA was removed by on-column DNase digestion (Qiagen) according to the manufacturer’s protocol. One microgram of RNA was used to synthesize cDNA using the high-capacity cDNA reverse transcription kit (Thermo Fisher Scientific) according to the manufacturer’s protocol. RT-qPCR analyses were performed using SsoFast EvaGreen supermix (Bio-Rad, Hercules, CA) according to the manufacturer’s protocol and GAPDH was used as a control. Primer sequences used for qRT-PCR are listed in Supplementary Table 2.