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Pipeline publication

[…] t RNA was isolated from 200 mg of total RNA by PEG 8000/NaCl precipitation. Small RNAs (20–30 nt) were purified from 15% denaturing PAGE gels and ligated first with the 5’ RNA adaptor and then with the 3’ RNA adaptor provided by Illumina. In each step, the ligated product was PAGE-gel purified. After first-strand synthesis and 18 cycles of PCR amplification, the product was PAGE-gel purified and submitted for sequencing on an Illumina GAIIx at MACROGEN (Seoul, Korea; The raw data and miRNA profiles submitted to NCBI under accession no.GSE39882., In a pre-processing step, we masked low-complexity and poly (A/T) sequences, and removed reads less than 100 bp using the SeqClean program (downloaded from the Dana Farber Cancer Institute; For contig assembly, we clustered reads into groups using megablast with criteria of more than 30 bp alignments between reads and identities greater than 94%. We then applied CAP3 to assemble the clustered reads into contigs []., The overall process and integrated databases are represented in Figure . To analyze functional annotation for each transcript, we aligned contig and singlet sequences with a lower expect value of 1e-10 against plant non-redundant (NR) proteins downloaded from the NCBI database [] and against Arabidopsis proteins downloaded from TAIR (ver. 10; []. Based on the Arabidopsis annotation, we integrated pathway information referring to KEGG [], FunCat [], and plant ontology (PO) [,]. Domains were identified with hmmPfam in the InterProScan Package []. Gene ontology (GO) was also analyzed based on the hmmPfam annotation. To exploit functionally related genes among related species, we downloaded protein and CDS sequences of the Rosaceae family (apple, strawberry, and peach), from GDR (, []), and grape, from Grape Genome Browser (, []), and analyzed orthologs and paralogs with OrthoMCL []., The scheme used for identifying miRNAs is presented in Figure . We consulted Breakfield et. al. paper for miRNA prediction []. To get high-quality small-RNA (sRNA) reads, we removed poor quality reads and adaptor sequences (5’-ATCTCGTATGCCGTCTTCTGCTTG- […]

Pipeline specifications

Software tools SeqClean, BLASTN, CAP3, InterProScan
Databases FunCat