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Pipeline publication

[…] Approximately 35 μg of total RNA were used for cDNA synthesis and library construction. All libraries were sequenced using the Illumina HiSeq 2500 platform. Small RNAs were first isolated from total RNA using 6 % agarose gels, and then purified. A library was then constructed using the Multiplex Small RNA Library Prep Set for Illumina (NEB)., Reference maize genome sequences were downloaded from Gramene (B73 AGPv3, Protein sequences were downloaded from Uniprot ( ID mapping for DNA and protein sequence matching was accomplished using R (ver. 3.1.2). Statistical analyses were conducted using R. Gene annotation based on BLAST was performed using Blast2GO []. The blast E-value was set as 1 × 10−3, and the E-Value-Hit-Filter was set as 1 × 10−6 in Blast2GO., For RNA-Seq, all reads from each sample were aligned to the reference genome of maize using TopHat2 []. Briefly, reads were first aligned to the reference genome by using Bowtie [] to identify splice junctions between exons and then the aligned reads were subjected to Cufflinks [] to assemble those reads into sample-specific transcriptomes using alignment coordinates., For microRNA-Seq, high-quality reads were aligned to the GenBank and Rfam [] databases to remove ncRNAs, with the exception of miRNAs. miRNAs were identified using miRDeep2 software []. Target prediction for known and unknown miRNAs was performed as reported previously using TargetFinder software []. Briefly, miRNA sequences were matched to the reference mRNA FASTA sequences and potential targets were computationally predicted by the match/mismatch-scoring ratio. Only predicted targets with scores less than four were considered reasonable., Gene expression levels were computed and expressed as reads per Kb per million fragments (RPKM), which was defined as:\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ \mathrm{RPKM} = \mathrm{total}\ \mathrm{e […]

Pipeline specifications

Software tools Blast2GO, TopHat, Cufflinks, miRDeep, Targetfinder
Organisms Zea mays, Arabidopsis thaliana, Homo sapiens
Chemicals Ribonucleotides, Heterocyclic Compounds, 2-Ring, Purine Nucleotides, Sugar Alcohols, Inositol, Inositol Phosphates