title: RNA-seq of clubroot infected B. napus roots
design: 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.