Instrument: Illumina HiSeq 2500
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: cDNA
Layout: SINGLE
Construction protocol: For each RNA extraction, frozen leaves from three plants were mixed and grinded to a fine powder using mortar and pistil under liquid nitrogen. Total RNA was isolated using a rapid cetyltrimethylammonium bromide (CTAB)-based extraction method with minor modifications (Gambino et al., 2008). DNA was removed by RNase-Free DNase (Qiagen) digestion in solution and then purified using RNAeasy Mini Kit (Qiagen). Primers for leucoanthocyanidin reductase 1 gene were used to check all RNA preparations for genomic DNA contamination (Bogs et al., 2005). RNA quality was analyzed using Experion RNA StdSens Chips (BioRad) to verify intact ribosomal bands [rRNA ratio (25s/18s) = 1.8-2.0] and RNA Quality Indicator (RQI) values > 8. For 0 MPI samples, 2 ug of the extracted total RNA was used to make the library. For the mixed second time point (0.5-1.5 MPI) 0.66 ug of extracted RNA from 0.5 MPI, 1 MPI and 1.5 MPI were mixed to give a total of 2ug RNA which was used to make the library. RNA extraction and library preparation was performed on biological triplicates for each of the four treatment time combination (IW 0 MPI, IW 0.5-1.5 MPI, NIW 0 MPI, NIW 0.5-1.5 MPI) to give a total of 12 libraries for RNA-Seq. RNA libraries for sequencing were prepared from 2ug of total RNA using a modified version of the Illumina TruSeq RNA Library Prep Protocol which is accessible at the website of the DNA Technologie Core, UC Davis (http://dnatech.genomecenter.ucdavis.edu/wp-content/uploads/2013/11/TruSeq_RNA_Sample_Prep_Sept2012.pdf). TruSeq Adapter used were AGTCAA (TruSeq Adapter, Index 13) for IW_0_MPI_1, AGTTCC (TruSeq Adapter, Index 14) for IW_0_MPI_2, ATGTCA (TruSeq Adapter, Index 15) for IW_0_MPI_3, CCGTCC (TruSeq Adapter, Index 16) for IW_0.5-1.5_MPI_1, GTCCGC (TruSeq Adapter, Index 18) for IW_0.5-1.5_MPI_2, GTGAAA (TruSeq Adapter, Index 19) for IW_0.5-1.5_MPI_3, GTGGCC (TruSeq Adapter, Index 20) for NIW_0_MPI_1, GTTTCG (TruSeq Adapter, Index 21) for NIW_0_MPI_2, CGTACG (TruSeq Adapter, Index 22) for NIW_0_MPI_3, GAGTGG (TruSeq Adapter, Index 23) for NIW_0.5-1.5_MPI_1, ACTGAT (TruSeq Adapter, Index 25) for NIW_0.5-1.5_MPI_2, ATTCCT (TruSeq Adapter, Index 27) for NIW_0.5-1.5_MPI_3. The 12 libraries were pooled, sequenced on two lanes of the same flow cell of a HiSeq2500 and de-multiplexed to produce 24 raw fastq files. TruSeq Adapter Index sequences are incorporated in the names of the raw fastq files.