Computational protocol: MiR-21 Is under Control of STAT5 but Is Dispensable for Mammary Development and Lactation

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Protocol publication

[…] RNA was prepared for sequencing using the TruSeq RNA Sample Prep Kit, Set A (Illumina catalog # FC-122-1001) according to manufacturer's recommendations. Each sample was prepared from 1 µg total RNA. cDNA was synthesized using random hexamer primers and SuperScript II (Invitrogen). The resulting cDNA libraries were quantified using the QuantiFluor™-ST system and checked for quality and size using the Agilent 2100 Bioanalyzer (High Sensitivity DNA kit, Agilent catalog # 5067-4626). A portion of each library was diluted to 10 nM and stored at −20°C. 2 µl of the 10 nM libraries were diluted and denatured, according to instructions from Illumina. The samples were loaded onto the cBot for clustering on a flow cell according to manufacturer's instructions (Illumina Inc., San Diego, CA) and sequenced using HiSeq 2000 (Illumina). The single-end sequenced tags from biological replicates of both wild-type and miR-21 −/− samples were aligned to the mouse reference genome (mm9 assembly) using the TopHat program . Transcript abundances were estimated by means of FPKM (fragment per kilobase of exon per million fragments mapped) and differentially expressed genes (DEGs) were initially identified by using the Cufflinks program . To remove potential false-positives, we only regarded the identified genes showing more than 5 FPKM in either miR-21+/+ or miR-21−/− samples as reliable DEGs. All statistical analyses were performed using a two-tailed unpaired Student's t test. A p-value of 0.05 or less was considered significant. The RNA-seq data are deposited in GEO under accession number (GSE50068). […]

Pipeline specifications

Software tools TopHat, Cufflinks
Application RNA-seq analysis
Organisms Mus musculus