mice were mated, grown and kept under standard conditions
Testes were dissected from male mice washed in and kept until processing in cold phosphate buffered saline solution. Miwi-associated small RNAs were isolated from mouse testes and libraries prepared by sequential ligation of 5’- and 3’- RNA adapters. Sequencing was for 36 cycles using the Illumina Genome Analyzer IIx. For RNAseq of round spermatids total RNA was depleted of abundant RNAs like rRNAs using Ribo-Zero kit (Epicentre). Strand-specific RNAseq library was prepared using the ScriptSeq mRNA-seq Library Preparation Kit (Epicentre; Cat no. SS10906). Sequencing was for 76 cycles by the Illumina Genome Analyzer. For global 5’-RACE library, total RNA from purified round spermatids was depleted of rRNAs using the Ribo-Zero kit (Epicentre). Then Illumina 5’ RNA adapter was ligated to capture pre-existing 5’ ends. After purification using the Absolutely RNA kit (Stratagene), RNA was reverse-transcribed with a random primer and PCR amplified. PCR products of ~200 bp were gel-eluted for sequencing by the Illumina Genome Analyzer (105 cycles). Most libraries are run in single lanes. Only one is duplexed and barcoded.
Illumina Genome Analyzer IIx
Adult-WT Small RNA Miwi IP
Barcodes where removed, and trimmed reads were mapped to the mouse genome mm9. The software used for processing the data (genomic coordinates etc) from the raw data files are in-house tools from Sachidanandam lab. They are described in a publication: Olson, A.J., Brennecke, J., Aravin, A.A., Hannon, G.J. & Sachidanandam, R. Analysis of large-scale sequencing of small RNAs. Pac Symp Biocomput, 126-36 (2008).