Small RNA-seq of Eucalyptus grandis: Unbent stem
Stem segments were macerated in liquid N2 and total RNA was extracted from 100 mg of ground stem tissue using CTAB and -mercaptoethanol and heated at 65C for 15 min. Chloroform:isoamyl alcohol (24:1) was added to the solution and it was centrifuged for 10min at 12000g. The supernatant was collected and precipitated with 2 volumes of absolute ethanol for 30 min at -80C. This solution was centrifuged for 20 min at -4C and 12000g; the pellet was resuspended in 50L of H2O. Total RNA was sent to Fasteris (Switzerland) for sRNA library construction and deep sequencing. The sRNA libraries were generated using Illumina TruSeq small RNA library prep kit. Small RNA libraries were multiplexed and sequenced in one lane using Illumina HiSeq. 2500 platform, generating 50bp single-end reads.