The plants were grown from October to December when the time of anthesis was determined for each head based on the dissection Paclitaxel microtubule of the middle spikelet. Immature grains were harvested from the middle six rows of the head at 1 to 15 Days Post An thesis. Total RNA was extracted from 100 mg of seeds from each DPA using the following method. Whole caryopsis was ground in a mortar using liquid nitrogen. 1. 2 mL of NTES buffer and 1. 6 mL of phenol chloroform isoamyl alcohol were added in the mortar and grinding continued until tissue was thawed. The extract was centrifuged 5 min at 12,000 rpm. The supernatant was precipitated by adding 1 10 vol of 3 M NaOAc and 2. 5 vol Inhibitors,Modulators,Libraries of 100% ethanol and incubating at ?20 C overnight. The extract was centri fuged 20 min at 4 C and 12,000 rpm.
The pellet was washed in 75% ethanol, centrifuged 5 min at maximum speed, Inhibitors,Modulators,Libraries dried for 1 min at room temperature and resus pended into 50 ul of RNAse free water. Inhibitors,Modulators,Libraries Samples were DNase treated using RQ1 DNase from Promega for 20 min at 37 C followed by a phenol chloroform extrac tion and a second ethanol Inhibitors,Modulators,Libraries precipitation. Total RNA extracts were resuspended into 50 uL of RNAse free water and the quality was determined using a Nanodrop spectrophotometer and agarose gel electrophoresis. An equal quantity of each RNA extract was pooled to consti tute the following three samples A Construction and analysis of smRNA libraries To determine the smRNA populations present in sam ples A, B and C, 60 ug of total RNAs were used to prepare libraries for Illumina sequencing.
A custom script was used to trim reads of 3 adapter sequences and then to pool identical reads to create a non redundant sequences list. Inhibitors,Modulators,Libraries Sequences that were over 50% homopoly mer or dinucleotide repeat were filtered out. Read counts for each sequence were normalised to reads per million of total sequenced. The diversity first of the signatures present in the three libraries had a high number of singletons, over 80%. To facilitate the analysis and build a level of confidence from the sequences cloned in the libraries, signatures were only considered that were 18 to 25 nucleotides in length and with a minimum expression of 1 RPM, represent ing 137,614 smRNAs. For identification of previously known miRNAs, signatures were checked for an exact match to a known miRNA present in miRBase or recently in barley leaves. To identify new miRNAs, the signatures of 19 to 23 nucleotides in length were kept and aligned to the HarvEST unigene set using SOAP. Signatures with no more than 20 matching unigenes and with no match to a smear of overlapping smRNA sequences were kept. For near identical sequences aligning to the same loca tion only the sequence with the highest read count was retained.