The smRNA signature was required to have no more than 4 mismatches against the complementary sequence in the hairpin structure and no more than 2 bulges. Con sidering that MIR genes may originate from the both evolu tion of an inverted repeat element that initially can produce endogenous 24 nt siRNAs, we kept the precursors sharing less than 70% homology within the pre miRNA region to repeat elements in the Plant Repeat Databases. smRNA which could be found in a stem of a potential miRNA precursor Inhibitors,Modulators,Libraries like hairpin structure in the folded sequences were marked as new miRNAs. For the downstream analysis of miRNA tar get genes, the remaining 19 23 nt signatures with no more than 90% homology to a repeat element were kept. Construction and analysis of degradome libraries The mRNA degradome libraries were made as described by German et al.
using total RNA extract from the 3 samples A, B and C. In brief, for each sample, 200 ug of total Inhibitors,Modulators,Libraries RNA was used to purify messenger RNA using an mRNA purification ki was linked to the mRNA 5CAP less fragments Inhibitors,Modulators,Libraries and purified again using the mRNA purification kit. After reverse transcrip tion using the RT primer, the cDNAs were amplified through 7 PCR cycles using the primers Amplicons were digested by MmeI and depho sphorylated by Shrimp Alkaline Phosphatase treatment. Samples were run on a 12% polyacrylamide gel and the MmeI cleaved fragments corresponding to the 42 bp gel band were purified. Purified Products were ligated to a double stranded DNA adaptor and purified again on 12 % polyacrylamide gel by excising the 86 bp band.
The purified amplicons, which constitute the degra dome libraries, were sequenced using the Illumina platform. As for the small RNA analysis, reads were trimmed, reduced to a non redundant set, filtered for repetitive se quence and their Inhibitors,Modulators,Libraries read counts were normalised in read per million. For subsequent analysis, sequences of 21 nt in length were trimmed back to 20 nt, then only sequences of 20 nt in length and having at least 1 RPM were retained. Kanga was used to align the 20 nt signatures to the HarvEST unigene sequences No mismatches were allowed in the alignment. For each matching EST the number of aligned degradome sequences at each position Inhibitors,Modulators,Libraries along the EST was investigated to identify signature peaks. Positions for which the number of aligned sequences exceeded the mean plus two standard devia tions for a sample along an EST were retained.
From each of these retained signature peaks, a 32 nt sequence was extracted from the EST, centred around the 5 end of the aligned signature, to constitute the Target Signature Sequence. To identify the smRNAs that could potentially selleck chemicals bind to a TSS we used psRNAtarget We ran the known miRNAs, new miRNAs and 19 23 nt smRNAs against the TSS with a maximum expectation of 5 and an hspsize equal to the length of the smRNA.