Hiseq 1000 sequencer

Total RNA was extracted using the Spectrum™ Plant Total RNA extraction kit (Sigma Aldrich) starting from 100 mg of deseeded berries, and total RNA yield was determined using a NanoDrop spectrophotometer (Thermo Fisher Scientific). Integrity of RNA samples was assessed on an RNA 6000 Nano Labchip using a Bioanalyzer 1000 (Agilent Technologies, Santa Clara, USA) prior to library preparation. Only samples showing a RIN (RNA Integrity Number) value higher than 8 were submitted to sequencing and quantitative expression analyses. The cDNA libraries (81 libraries in total, Supplementary Table S3) were prepared from samples collected in 2013 using TruSeq RNA Sample Prep Kit v2 (Illumina, San Diego, USA), starting from 2.5 µg of total RNA and following the manufacturer’s instructions. Libraries were sequenced with an Illumina HiSeq 1000 sequencer (Illumina Inc., San Diego, CA, USA) generating ∼23 million 100 bp single-end reads per sample (Supplementary Table S3). Low-quality reads (> 50 bases with quality < 7 or > 10% undetermined bases) were removed and Illumina TruSeq adapter sequences were clipped.

RNA was extracted from three independent biological replicates of each culture condition at exponential phase using a modified TRIzol SIGMA-ALDRICH protocol. Cells were disrupted by glass beads (Micro-organism lysing VKmix, Bertin Technologies). RNA samples were further purified with the SIGMA Spectrum Plant Total RNA kit including a DNAse treatment step. RNA quality and quantity were determined using a Nanodrop 2000 spectrophotometer (Thermo Scientific, Wilmington, DE) and a Bioanalyzer Chip RNA 7500 series II (Agilent, Santa Clara, CA). Directional RNA-seq library preparation was performed starting from 1 ug total RNA using the TruSeq RNA Sample Prep Kit v2 (Illumina Inc., San Diego, CA, USA) after capturing poly-adenylated transcripts. Library quality was assessed with a High Sensitivity DNA Kit on a 2200 Tape Station (Agilent, Wokingham, UK). Libraries were sequenced with an Illumina HiSeq. 1000 sequencer (Illumina Inc., San Diego, CA, USA) generating ~22 million 100-bp paired-end reads per sample. Low-quality reads (>50 bases with quality <7 or >10% undetermined bases) and putative PCR duplicate reads were removed and Illumina TruSeq adapter sequences were clipped. Low-quality bases at read ends were trimmed (minimum quality 16, minimum read length 50 bp) with cutadapt (http://code.google.com/p/cutadapt/).

Groups of randomly-selected plants (3 for each cultivar) per treatment (inoculated and non-inoculated) and time point (see above) were defined in order to reduce plant-to-plant variability, as described by Leyva-Pérez and co-workers [25 (link)]. Total RNA samples from roots sampled at the time-points indicated above were extracted using a ‘Spectrum Plant Total RNA kit’ (Sigma-Aldrich, St Louis, MO, USA), following the manufacturer’s indications. Contaminant DNA was removed with on-column DNase digestion (Roche, Basel, Switzerland). The Agilent 2100 bioanalyser (Agilent Technologies, Santa Clara, CA, USA) using an RNA 6000 Pico assay kit (Agilent Technologies) was employed for RNA quality tests. Then, equimolar amounts of RNA from each group were pooled, cDNA libraries were prepared, and NGS sequencing was performed at Sistemas Genómicos S.L. (Paterna, Valencia, Spain) using an Illumina HiSeq 1000 sequencer. Two replicates per sample were sequenced on different lanes in the flow cell [25 (link)].

Small RNA libraries were prepared according to Small RNA v1.5 sample preparation guide (Illumina, January 2010). Briefly, 5 μg of total RNA is used for the small library preparation. Adapters are added at each ends of the small by ligation. First, the ligation is made at the 3′end and then a second ligation is made at the 5′end. v1.5 small RNA 3′ adapter is specifically modified to target miRNAs and other small RNAs that have a 3′ hydroxyl group resulting from enzymatic cleavage by Dicer or other processing enzymes. Small RNAs with both adapters are reverse-transcribed and amplified by PCR. Amplified small RNA libraries are gel purified on a polyacrylamide gel (miRNAs are 89 nt to 96 nt long with both adapters). DNA quality and integrity were checked using DNA-1000 chip (Agilent).
The small library is then hybridized on the flow cell and the libraries’ clustering was performed with the C-Boot cluster machine. The sequencing was done using a HiSeq-1000 sequencer (Illumina).

We used our recently published strategy to generate 3′-seq libraries and the computational workflow to analyze the data [29 (link)]. Briefly, 3′-seq libraries were prepared using 2 μg of total RNA as starting material. The total RNA was chemically fragmented and custom oligo-dT primers were used to capture and synthesize cDNA representing the junction of the poly(A) tail and the 3′ end of RNAs. The cDNAs were sequenced using an Illumina HiSeq-1000 sequencer with SE-50 mode. The data were mapped onto the genome assemblies and 3′ end clusters were derived and quantified within a 25-bp window as described [29 (link)].