title: GSM2881243: MIWI2 IP;P0;Tex15-/- [RR564]; Mus musculus; RIP-Seq
library_construction_protocol: Total small RNAs were isolated from E16.5 embryos. Briefly, total RNA from E16.5 testes was separated on 15% UREA polyacrylamide gel. The gel was cut between 16-40 nts to isolate total small RNAs. RNAs present in endogenous MILI, and MIWI2, complexes were isolated. Briefly, immunoprecipitations were treated with Proteinase K in 300 μl reaction at 42 °C for 15 min (10 mM Tris-HCl pH 7.5, 5 mM EDTA, 0.5% SDS). RNAs present in the sample were purified by phenol-chloroform extraction and precipitated with ethanol. Approximately 10-20% of the sample was labelled at the 5′ end with polynucleotide kinase (PNK, ThermoFisher Scientific) and [γ 32P] ATP (3000 Ci/mmol, Perkin Elmer), and resolved by 15% urea-PAGE for quality check. Libraries were prepared (barcoded at 3′ end) using NEBNext® Multiplex Small RNA Library Prep Set for Illumina® (NEB Catalogue No. E7300) following manufacturer instructions. The synthesized cDNA libraries were resolved on 3% high-resolution MethaPhor agarose (Lonza, cat. No. 50180) gels in TAE buffer. Fragments in the size-range of ~160 bp were gel-extracted with the use of MinElute Gel Extraction Kit (Qiagen, cat No. 28604). Multiple libraries with different barcodes (at 3′ end) were mixed in equimolar ratios and sequenced with the Illumina HiSeq 2000 platform (EMBL GeneCore facility, Heidelberg).
attributes.tag: GEO Accession
attributes.value: GSM2881243