RIP-seq and RIP-qPCR analysis

RIP-seq was performed as described previously with minor modifications^58–60. Briefly, RIP was performed using the complementary strain expressing FLAG-tagged FgRbp1 in ΔFgRbp1 (ΔFgRbp1::FgRbp1-FLAG). Fresh mycelia samples were harvested as described earlier in RNA-seq methodology. Fresh mycelia were ground into fine powder in liquid nitrogen and suspended with 4 mL lysis buffer (50 mM Tris-HCl [pH 7.5], 150 mM KCl, 2 mM EDTA, 0.5% NP-40, 0.5 mM dithiothreitol, 1:100 v/v protease inhibitor cocktail [Sangon Biotech, Shanghai, China], 200 units/ml RNase OUT [Invitrogen]), followed by simple sonication (30 s on, 30 s off, repeated twice). Homogenates were centrifuged for 20 min at 16000 × g under 4 °C to clear the lysate, and a 200-μl sample was saved as the input sample. The remaining sample was divided into two equal parts (IP sample and mock sample) and incubated with anti-Flag M2 (Sigma, F1804) antibody (IP sample antibody) or anti-IgG1 (Thermo Scientific, MA1-10406) antibody (mock sample antibody) together with the magnetic protein G beads (Life Technologies; 10004D) at 4 °C overnight with rotation. After incubation, the bead-protein-RNA complexes were washed with ice-cold NT2 buffer (50 mM Tris-HCl (pH 7.4), 1 mM MgCl₂, 150 mM NaCl, 0.05% NP40, 0.5 mM dithiothreitol, 1:100 v/v protease inhibitor cocktail and 200 units/ml RNase OUT) for five times. After the last washing step, the beads were treated with 30 μg proteinase K at 55 °C for 30 min to release the RNP complexes and RNA was then extracted with Trizol reagent. IP and input RNAs were shipped to Novogene for library construction and sequencing on Illumina HiSeq 2500 (Novogene, Beijing, China) or used for quantitative PCR analysis.

For RIP-qPCR, RNAs from input, IP and mock sample were reverse transcribed into cDNA and subjected to qRT-PCR using SYBR green I fluorescent dye detection. Primer sequences are described in Supplementary Data ⁵. Fold enrichment of tested genes was calculated using the equation 2^{Δ(Ct(mock-input) – Ct(IP-input))}. Meanwhile, ACTIN was also amplified as a negative control. For every analyzed RNA fragment, each sample was quantified from three independent RIP analyses.