title: RNA-seq of ectomycorrhizal fungi
design: A total amount of 1.5 mg total RNA per mycelium sample was used as input for RNA sample preparation and three repetitions per treatment. Sequencing libraries were generated using an NEBNext Ultra RNA library prep kit for Illumina (NEB, USA) following the manufacturer's recommendations, and index codes were added to attribute sequences to each sample. Briefly, mRNA was purified from total RNA using polyT oligo-attached magnetic beads. Fragmentation was conducted using divalent cations under elevated temperature in NEBNext first strand synthesis reaction buffer (5). First-strand cDNA was synthesized using random hexamer primers and M-MuLV reverse transcriptase (RNase H-). Second-strand cDNA synthesis was subsequently performed using DNA polymerase I. The remaining overhangs were converted into blunt ends using exonuclease/polymerase activities. After adenylation of the 3 ends of the DNA fragments, NEBNext adaptors with hairpin loop structures were ligated to prepare for hybridization. To select cDNA fragments of preferentially 150-200 bp in length, the library fragments were purified with an AMPure XP system (Beckman Coulter, Beverly, USA). Then, 3 L of USER enzyme (NEB, USA) was used with size-selected, adaptor-ligated cDNA at 37  for 15 min followed by 5 min at 95 before PCR. Then, PCR was performed with Phusion High-Fidelity DNA polymerase, universal PCR primers, and an Index (X) primer. Finally, PCR products were purified (AMPure XP system), and library quality was assessed on the Agilent Bioanalyzer 2100 system. After the library quality inspection qualified, we performed the paired-end sequencing on an Illumina Hiseq 2500 at the (lc-bio, China) following the vendor's recommended protocol.