Computational protocol: Cellular and molecular characterization of gametogenic progression in ex vivo cultured prepuberal mouse testes

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Protocol publication

[…] RNA size separation, preparation of libraries and NGS were performed by a commercial agreement with the Beijing Genomics Institute (China) using 2 μg of total RNA isolated from each sample using the HiSeq 2000 (Illumina, California, USA). Small RNA-seq was performed at a depth of 10 million sequences and a 50 nucleotide extension. Thereafter used adaptors were trimmed and a read quality analysis using the program FastQC was assessed (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). Once the data had been filtered, reads were aligned against the mouse genome (mm10) using the Bowtie aligner []. Next, reads were sequentially aligned against different sncRNAs databases retaining the mapped reads: miRBase 21 [], piRBase [#1], and Ensembl’s non-coding RNAs database [#2]. Finally, reads that did not map against any of the sncRNAs databases nor against the mouse genome were classified as “unannotated”. The bioinformatic pipeline is included in the Additional File . […]

Pipeline specifications

Software tools FastQC, Bowtie
Databases miRBase piRBase
Application sRNA-seq analysis
Organisms Mus musculus