mRNA-seq of Arabidopsis rosette: stress treatment Drought 9 days rep  3
RNA was isolated using Trizol Reagent (Life Technologies Australia Pty Ltd) using a procedure adapted from Allen et al. (2010). The Illumina TruSeq V2 kit was used for RNA-seq library preparation as per the manufacturer's instructions (TruSeq User Guide Revision D) with a custom dUTP incorporation step during second strand synthesis to introduce strand-specificity (adapted from Parkhomchuk et al. 2009).  Four micrograms of total RNA were used for input and fragmented for 8 minutes. Following first strand synthesis, cDNA was precipitated to remove dNTPs; per 25 ll first strand reaction 5 ll of 5M NH4OAc (ammonium discriminates against precipitation of dNTPs), 2 ll of the co-precipitant Glycolblue (Ambion) and 110 ll 100% ethanol were added and samples incubated overnight at -80çC. The following day samples were recovered by centrifugation at 4çC, pellets washed with 70% ethanol, air-dried for 5 minutes then resuspended in custom second strand buffer incorporating a dNTP/dUPT mix.  The second strand mix contained, 4 ll of 5x RT buffer from the SuperScript III kit (Life Technologies), 2 ll 50 mM MgCl2, 2 ll 0.1 M DTT, 1 ll 50 ng/ll random hexamers, 49 ll H2O, 8 ll Second Strand Synthesis buffer (NEBNext, B6117S), 10 ll dNTP/dUTP mix, (Fermentas, R0251), 4 ll Second Strand enzyme mix (NEBNext, E6111S) and samples were incubated for 2.5 hours at 16çC. Prior to the PCR enrichment step (after adapter ligation), the USER enzyme was used to degrade the dUTP containing coding strand to give a reverse stranded final library. Per sample, 1 ll of USER enzyme mix was added and samples incubated at 37çC for 15 minutes.  Using pilot PCRs an optimal PCR cycle number of 15 cycles was determined empirically for amplification of all samples and final amplification was performed with a half volume PCR. Following PCR clean up samples were pooled in equal molar ratios and sequenced (100bp paired-end) on one lane of the HiSeq2000.