Computational protocol: Transcription of the Major Neurospora crassa microRNA–Like Small RNAs Relies on RNA Polymerase III

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Protocol publication

[…] Three independent wild-type cultures grown in liquid media were pooled for RNA isolation for sequencing. RNA sequencing and data analyses were performed by Beijing Genomic Institute in Shenzhen, China. Strand specific sequencing was carried out following the method introduced by Dmitri Parkhomchuk . mRNAs that purified from total RNA were sheared and reverse-transcribed into first-strand cDNAs. dTTP was substituted by dUTP during the synthesis of the second-strand cDNAs. After cDNA fragmentation and adaptor ligation, the dUTP containing second strand population were removed by Uracil-N-Glycosylase. The resulting first strand cDNA population were sequenced by Illumina HiSeq 2000. The cleaned reads were assembled and mapped to the reference Neurospora genome sequence ( using SOAP (Short Oligonucleotide Alignment Program) , tophat , and cufflinks –. The strand-specific results were displayed and analyzed by IGV (Integrative Genomics Viewer) software . […]

Pipeline specifications

Software tools SOAP, TopHat, Cufflinks, IGV
Application RNA-seq analysis
Organisms Neurospora crassa
Diseases Mycoses