title: GSM1650026: RIP_Ruvbl1_rep1; Mus musculus; RIP-Seq
library_construction_protocol: Cells were lysed using an NE-PER Extraction kit (Thermo Fisher) to isolate cytoplasmic and nuclear fractions. For immunoprecipitation, 1.5 mg of nuclear extracts were treated with DNase I (New England Biolabs) and pre-cleared with Protein A magnetic beads (New England Biolabs) for 3 hours. Cleared nuclear extract was incubated with specific antibodies in IP buffer (50 mM Tris-HCl pH7.4, 250 mM NaCl, 0.1% Triton X-100), plus 1X HALT protease inhibitors (Thermo Fisher) and SUPERaseIn (Life Technologies) overnight at 4°C. The next day, pre-washed Protein A magnetic beads were added to IP samples and incubated for another 4 hours at 4°C. The magnetic beads were sequentially washed with IP buffer twice, high-salt IP buffer (50 mM Tris-HCl pH7.4, 500 mM NaCl, 0.1% Triton X-100, 0.5 % sodium deocycholate) four times, and IP buffer two more times. RNA was eluted from beads and purified by TRIzol (Life Technologies) extraction and precipitated at -80°C for at least 2 hours. 10-50 ng of RIP enriched RNA and Adaptor 1 (5’-CTGAACCGCTCTTCCGATCTNNNNNN-3’) were used for first-strand cDNA synthesis with Superscript III Reverse Transcription Kit (Life Technologies). After first-strand cDNA synthesis, RNA was degraded by sodium hydroxide and cDNA was purified by SILAN beads (Life Technologies). To preserve strand information, Adaptor 2 with the modification of 5’ phosphorylated and 3’ dideoxy-C (5’-p-NNACGTAGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT-3’ddC) was ligated to the 3’ end of first-strand cDNA using T4 RNA ligase 1 (New England Biolabs). The ligated material was purified by SILAN beads and PCR amplified with Illumina primers using 18 cycles of PCR. To remove PCR primers, libraries were purified by AMPure XP beads (Beckman Coulter).