GSM3103097: P0 Ovary Input RNA; Mus musculus; RIP-Seq
One hundred newborn ovaries were homogenized in IP buffer (20 mM HEPES/KOH, pH7.5, 150 mM NaCl, 2.5 mM MgCl2, 0.1% NP-40, 1 mM dithiothreitol, 1× protease inhibitor cocktail (Roche), 100 U ml-1 RNase inhibitor (Takara)),and the debris was removed by centrifugation (10000 × g at 4°C for 10 min).The supernatants were incubated with magnetic beads conjugated to protein G (Invitrogen), which were pre-incubated with anti-ELAVL2 antibody (5 μg, Protein tech, 14008-1-AP) at 4°C for 2 hours.The lysate-bead mixtures were then incubated at 4°C for 6 hours with gentle rotation. The bead-antibody complexes were washed thrice with IP buffer and transferred to fresh 1.5 ml tubes. The complexes were washed again with IP buffer. The precipitated proteins and RNA were eluted by incubating the beads-antibody complex with the elution buffer (IP buffer containing 0.5% SDS) at 70°C for 5 min. Input samples (5% of starting materials) were taken from the centrifuged supernatants. Input and eluted samples were dissolved in TRIzol reagent (Invitrogen), and total RNAs were extracted according to the manufacture's protocol. The quality of RNAs was checked using the 2100 Bioanalyzer (Agilent). Anti-sense RNAs were amplified by using TargetAmp 1-Round aRNA Amplification Kit 103 (Epicentre) according to the manufacture's protocol, and sequence libraries were then generated using the KAPA Stranded mRNA-Seq Kit Illumina platform (KAPA Biosystems) according to the manufacturer's protocol.  The resulting libraries were sequenced on the Illumina GAIIx (single end 36 base reads with TruSeq SBS kit v5 for GA., Illumina).