sRNA-seq of Arabidopsis rosette: RRGD Timepoint VI - recovery 15 min rep  3
RNA was isolated using Trizol Reagent (Life Technologies Australia Pty Ltd) using a procedure adapted from Allen et al. (2010). Small RNAs were size fractionated from 10 lg of total RNA using PAGE on 15%, 8 M TBU gels, by excising the region from approximately 18-35 bp. Sequencing libraries were prepared using the NEB Next sRNA kit (E7300), according to the manufactureres instructions from 5 lg of isolated sRNA. Ligations were performed for 1 hr and pilot PCRs (20 ll) were performed on the cDNA to determine an optimal PCR cycle number of 13, and final PCR was performed using a 40 ll reaction in duplicate. Amplified libraries were size selected by PAGE on 6% 0.5 x TBE gels. Libraries were prepared in biological triplicate, indexed, pooled and sequenced over 2 lanes of the Illumina HiSeq2000 in 50bp single end mode.