Seedlings were grown on media containing 1 xMS salts, 1% sucrose. 2-week-old seedlings were sprayed with water. After 2 hours, seedlings were collected and total RNA was isolated using TRIzol reagent.
Data processing
To determine the normalized abundance value for each signature in the library, we separately merged 2-step runs and 3-step runs to create a raw abundance count for each stepper. The raw abundance count for each stepper was calculated as the average from all 2-step or from all 3-step runs for a given signature. The average total number of signatures sequenced in all 2-step or for all 3-step runs was also calculated within the library. The final stage to merge the data within the steppers was to calculate the normalized abundance for each stepper. The normalized abundance is the raw abundance count divided by the average total number of signatures for both of the steppers in the library, multiplied by 250,000 to obtain a "transcripts per quarter million" or TPQ value. For each signature, only the one normalized value is used - either the 2-step or 3-step normalized value, selected based on the higher of the sum of the 2-step or 3-step data across all libraries. More details are available in Meyers et al. (2004, Genome Research 14:1641) or Lu et al. (2005). This method was applied separately for signatures matching to tRNAs, rRNAs, snoRNAs or snRNAs. These signatures were removed from the primary set of small RNA signatures and treated as a separate set.
