RNA-seq of clubroot infected B. napus roots
A total amount of 3 g RNA per sample was used as input material for the  RNA samplepreparations. Firstly, ribosomal RNA was removed by Epicentre  Ribo-zeroTM rRNA RemovalKit (Epicentre, USA), and rRNA free residue was  cleaned up by ethanol precipitation.Subsequently, sequencing libraries were  generated using the rRNA-depleted RNA by NEBNextUltraTM Directional RNA  Library Prep Kit for Illumina (NEB, USA) followingmanufacturers  recommendations. Briefly, fragmentation was carried out using divalent  cationsunder elevated temperature in NEBNext First Strand Synthesis Reaction  Buffer(5X). First strandcDNA was synthesized using random hexamer primer and  M-MuLV ReverseTranscriptase(RNaseH-). Second strand cDNA synthesis was  subsequently performed usingDNA Polymerase I and RNase H. In the reaction  buffer, dNTPs with dTTP were replaced bydUTP. Remaining overhangs were  converted into blunt ends via exonuclease/polymeraseactivities. After  adenylation of 3 ends of DNA fragments, NEBNext Adaptor with hairpinloop structure  were ligated to prepare for hybridization. In order to select cDNA fragments  ofpreferentially 150~200 bp in length, the library fragments were purified  with AMPure XP system(Beckman Coulter, Beverly, USA). Then 3 l USER Enzyme  (NEB,USA) was used with size selected,adaptor-ligated cDNA at 37C for 15 min  followed by 5 min at 95C before PCR. ThenPCR was performed with Phusion  High-Fidelity DNA polymerase, Universal PCR primers andIndex(X) Primer. At  last, products were purified (AMPure XP system) and library quality was  assessed onthe Agilent Bioanalyzer 2100 system.