title: Ryegrass plant with idtM minus endophyte with GA3 applied
design: The RNA extraction was performed on 50-100 mg of fresh ground samples using the Bioline ISOLATE II RNA Plant Kit (Meridian Bioscience, UK). RNA purity, concentration and integrity were determined with NanoPhotometer spectrophotometer (IMPLEN, USA), Qubit 2.0 fluorometer (Life Technologies, USA), and Agilent 2100 Bioanalyzer (RNA Nano 6000 assays, USA), respectively. A total of 300 ng of RNA was used for library preparation following the manufacturers protocol (VAHTS Universal V8 RNA-seq Library Prep Kit for Illumina). The poly(A) mRNA isolation was performed using VAHTS mRNA Capture Beads (Vazyme, China) or Ribo-MagOff rRNA Depletion Kit (Vazyme, China). The mRNA fragmentation and priming were performed using 2  Frag/Prime Buffer. First strand cDNAs were synthesized using 1st Strand Buffer 3/1st Strand Enzyme Mix 3. The synthesis of second strand cDNAs, and addition of end prepairs and dA-tailings were conducted using 2nd Strand Buffer 2/2nd Strand Enzyme Super Mix 2, followed by a T-A ligation. Size selection of Adaptor-ligated DNA was then performed using VAHTS DNA Clean Beads (Vazyme, China), and fragments of ~260-360 bp (with the approximate insert size of 200-300 bp) were recovered. Each sample was then amplified by PCR for 12-15 cycles using P5 and P7 primers. The PCR products were cleaned up with VAHTS DNA Clean Beads (Vazyme, China), and their quantity and quality were measured with Qubit 3.0 Fluorometer (Invitrogen, USA) and Agilent 4200 Bioanalyzer (Agilent Technologies, USA).