stage: adult size selection: ~18-30 nt antibody: none
Treatment protocol
Testes extract was produced by decapsulating testicles, washing and resuspending spermatogenic tubules in protein extraction buffer, then douncing tubules with 20 strokes of a Type B glass pestle. Extract was then cleared with a 30 minute, 10,000 g spin, and snap frozen.
Growth protocol
Mouse testes were dissected from euthanized adult male swiss webster mice.
Extracted molecule
total RNA
Extraction protocol
The anti-Mili or anti-Miwi antibody was immobilized onto magnetic beads, and beads were suspended in testes extract for 1 hour. Retained beads were washed 6 times with 1X PBS, then total RNA from the IP was extracted with Tri-reagent. Total RNA from whole testes extract was also extracted with Tri-reagent. Adapters were ligated to the small RNA component from the IP or total RNA, and a cDNA library was produced by reverse-transcription of the adapter-ligated small RNAs. The cDNA library was gel purified, quantitated, and coupled to a flow cell for sequencing on the Illumina Genome Analyzer II.
Library strategy
RNA-Seq
Library source
transcriptomic
Library selection
size fractionation
Instrument model
Illumina Genome Analyzer II
Description
total RNA IP and gel-purification, RNA-seq. Sequencing of small RNAs from Mili IP, Miwi IP, and total RNA from mouse adult testes extract.
Data processing
Raw sequences were clipped by 3' linker sequences recognition, and clipped sequences longer than 18 nt were selected for analysis. The processed data files (*seq ) are the result of the pipeline: Raw files -> remove the linker sequences -> count the number of each sequence.
