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
P14-WT Small RNA Miwi IP read_length: 36
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).